Microfabricated elastomeric valve and pump systems

ABSTRACT

A method of fabricating an elastomeric structure, comprising: forming a first elastomeric layer on top of a first micromachined mold, the first micromachined mold having a first raised protrusion which forms a first recess extending along a bottom surface of the first elastomeric layer; forming a second elastomeric layer on top of a second micromachined mold, the second micromachined mold having a second raised protrusion which forms a second recess extending along a bottom surface of the second elastomeric layer; bonding the bottom surface of the second elastomeric layer onto a top surface of the first elastomeric layer such that a control channel forms in the second recess between the first and second elastomeric layers; and positioning the first elastomeric layer on top of a planar substrate such that a flow channel forms in the first recess between the first elastomeric layer and the planar substrate.

CROSS-REFERENCES TO RELATED APPLICATIONS

This nonprovisional patent application is a continuation of U.S.application Ser. No. 14/188,664 filed Feb. 24, 2014, which is acontinuation of Ser. No. 11/932,263 filed Oct. 31, 2007, now U.S. Pat.No. 8,656,958, which is a continuation of U.S. application Ser. No.11/685,654 filed Mar. 13, 2007, now U.S. Pat. No. 8,104,497, which is acontinuation of U.S. application Ser. No. 11/056,451 filed Feb. 10,2005, now U.S. Pat. No. 7,216,671, which is a continuation of Ser. No.09/826,583 filed Apr. 6, 2001, now U.S. Pat. No. 6,899,137, which is acontinuation-in-part of U.S. application Ser. No. 09/724,784 filed Nov.28, 2000, now U.S. Pat. No. 7,144,616, which is a continuation-in-partof U.S. application Ser. No. 09/605,520 filed Jun. 27, 2000, now U.S.Pat. No. 7,601,270, which claims the benefit of U.S. Appl. No.60/141,503 filed Jun. 28, 1999; U.S. Appl. No. 60/147,199 filed Aug. 3,1999; and U.S. Appl. No. 60/186,856 filed Mar. 3, 2000, the disclosuresof which are incorporated by reference herein for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No.HG-01642-02 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

TECHNICAL FILED

The present invention relates to microfabricated structures and methodsfor producing microfabricated structures, and to microfabricated systemsfor regulating fluid-flow.

BACKGROUND OF THE INVENTION

Various approaches to designing micro-fluidic pumps and valves have beenattempted. Unfortunately, each of these approaches suffers from its ownlimitations.

The two most common methods of producing microelectromechanical (MEMS)structures such as pumps and valves are silicon-based bulkmicro-machining (which is a subtractive fabrication method wherebysingle crystal silicon is lithographically patterned and then etched toform three-dimensional structures), and surface micro-machining (whichis an additive method where layers of semiconductor-type materials suchas polysilicon, silicon nitride, silicon dioxide, and various metals aresequentially added and patterned to make three-dimensional structures).

A limitation of the first approach of silicon-based micro-machining isthat the stiffness of the semiconductor materials used necessitates highactuation forces, which in turn result in large and complex designs. Infact, both bulk and surface micro-machining methods are limited by thestiffness of the materials used. In addition, adhesion between variouslayers of the fabricated device is also a problem. For example, in bulkmicro-machining, wafer bonding techniques must be employed to createmultilayer structures. On the other hand, when surface micro-machining,thermal stresses between the various layers of the device limits thetotal device thickness, often to approximately 20 microns. Using eitherof the above methods, clean room fabrication and careful quality controlare required.

BRIEF SUMMARY OF THE INVENTION

The present invention sets forth systems for fabricating and operatingmicrofabricated structures such as on/off valves, switching valves, andpumps e.g. made out of various layers of elastomer bonded together. Thepresent structures and methods are ideally suited for controlling andchanneling fluid movement, but are not so limited.

In a preferred aspect, the present invention uses a multilayer softlithography process to build integrated (i.e.: monolithic)microfabricated elastomeric structures.

Advantages of fabricating the present structures by binding togetherlayers of soft elastomeric materials include the fact that the resultingdevices are reduced by more than two orders of magnitude in size ascompared to silicon-based devices. Further advantages of rapidprototyping, ease of fabrication, and biocompatability are alsoachieved.

In preferred aspects of the invention, separate elastomeric layers arefabricated on top of micromachined molds such that recesses are formedin each of the various elastomeric layers. By bonding these variouselastomeric layers together, the recesses extending along the variouselastomeric layers form flow channels and control lines through theresulting monolithic, integral elastomeric structure. In various aspectsof the invention, these flow channels and control lines which are formedin the elastomeric structure can be actuated to function as micro-pumpsand micro-valves, as will be explained.

In further optional aspects of the invention, the monolithic elastomericstructure is sealed onto the top of a planar substrate, with flowchannels being formed between the surface of the planar substrate andthe recesses which extend along the bottom surface of the elastomericstructure.

In one preferred aspect, the present monolithic elastomeric structuresare constructed by bonding together two separate layers of elastomerwith each layer first being separately cast from a micromachined mold.Preferably, the elastomer used is a two-component addition cure materialin which the bottom elastomeric layer has an excess of one component,while the top elastomeric layer has an excess of another component. Inan exemplary embodiment, the elastomer used is silicone rubber. Twolayers of elastomer are cured separately. Each layer is separately curedbefore the top layer is positioned on the bottom layer. The two layersare then bonded together. Each layer preferably has an excess of one ofthe two components, such that reactive molecules remain at the interfacebetween the layers. The top layer is assembled on top of the bottomlayer and heated. The two layers bond irreversibly such that thestrength of the interface approaches or equals the strength of the bulkelastomer. This creates a monolithic three-dimensional patternedstructure composed entirely of two layers of bonded together elastomer.Additional layers may be added by simply repeating the process, whereinnew layers, each having a layer of opposite “polarity” are cured, andthereby bonded together.

In a second preferred aspect, a first photoresist layer is deposited ontop of a first elastomeric layer. The first photoresist layer is thenpatterned to leave a line or pattern of lines of photoresist on the topsurface of the first elastomeric layer. Another layer of elastomer isthen added and cured, encapsulating the line or pattern of lines ofphotoresist. A second photoresist layer is added and patterned, andanother layer of elastomer added and cured, leaving line and patterns oflines of photoresist encapsulated in a monolithic elastomer structure.This process may be repeated to add more encapsulated patterns andelastomer layers. Thereafter, the photoresist is removed leaving flowchannel(s) and control line(s) in the spaces which had been occupied bythe photoresist. This process may be repeated to create elastomerstructures having a multitude of layers.

An advantage of patterning moderate sized features (>1=10 microns) usinga photoresist method is that a high resolution transparency film can beused as a contact mask. This allows a single researcher to design,print, pattern the mold, and create a new set of cast elastomer devices,typically all within 24 hours.

A further advantage of either above embodiment of the present inventionis that due to its monolithic or integral nature, (i.e., all the layersare composed of the same material) is that interlayer adhesion failuresand thermal stress problems are completely avoided.

Further advantages of the present invention's preferred use of asilicone rubber or elastomer such as RTV 615 manufactured by GeneralElectric, is that it is transparent to visible light, making amultilayer optical trains possible, thereby allowing opticalinterrogation of various channels or chambers in the microfluidicdevice. As appropriately shaped elastomer layers can serve as lenses andoptical elements, bonding of layers allows the creation of multilayeroptical trains. In addition, GE RTV 615 elastomer is biocompatible.Being soft, closed valves form a good seal even if there are smallparticulates in the flow channel. Silicone rubber is also bio-compatibleand inexpensive, especially when compared with a single crystal silicon.

Monolithic elastomeric valves and pumps also avoid many of the practicalproblems affecting flow systems based on electro-osmotic flow.Typically, electro-osmotic flow systems suffer from bubble formationaround the electrodes and the flow is strongly dependent on thecomposition of the flow medium. Bubble formation seriously restricts theuse of electro-osmotic flow in microfluidic devices, making it difficultto construct functioning integrated devices. The magnitude of flow andeven its direction typically depends in a complex fashion on ionicstrength and type, the presence of surfactants and the charge on thewalls of the flow channel. Moreover, since electrolysis is taking placecontinuously, the eventual capacity of buffer to resist pH changes mayalso be reached. Furthermore, electro-osmotic flow always occurs incompetition with electrophoresis. As different molecules may havedifferent electrophoretic mobilities, unwanted electrophoreticseparation may occur in the electro-osmotic flow. Finally,electro-osmotic flow cannot easily be used to stop flow, halt diffusion,or to balance pressure differences.

A further advantage of the present monolithic elastomeric valve and pumpstructures are that they can be actuated at very high speeds. Forexample, the present inventors have achieved a response time for a valvewith aqueous solution therein on the order of one millisecond, such thatthe valve opens and closes at speeds approaching or exceeding 100 Hz. Inparticular, a non-exclusive list of ranges of cycling speeds for theopening and closing of the valve structure include between about 0.001and 10000 ms, between about 0.01 and 1000 ms, between about 0.1 and 100ms, and between about 1 and 10 ms. The cycling speeds depend upon thecomposition and structure of a valve used for a particular applicationand the method of actuation, and thus cycling speeds outside of thelisted ranges would fall within the scope of the present invention.

Further advantages of the present pumps and valves are that their smallsize makes them fast and their softness contributes to making themdurable. Moreover, as they close linearly with differential appliedpressure, this linear relationship allows fluid metering and valveclosing in spite of high back pressures.

In various aspects of the invention, a plurality of flow channels passthrough the elastomeric structure with a second flow channel extendingacross and above a first flow channel. In this aspect of the invention,a thin membrane of elastomer separates the first and second flowchannels. As will be explained, downward movement of this membrane (dueto the second flow channel being pressurized or the membrane beingotherwise actuated) will cut off flow passing through the lower flowchannel.

In optional preferred aspects of the present systems, a plurality ofindividually addressable valves are formed connected together in anelastomeric structure and are then activated in sequence such thatperistaltic pumping is achieved. More complex systems includingnetworked or multiplexed control systems, selectably addressable valvesdisposed in a grid of valves, networked or multiplexed reaction chambersystems and biopolymer synthesis systems are also described.

One embodiment of a microfabricated elastomeric structure in accordancewith the present invention comprises an elastomeric block formed withfirst and second microfabricated recesses therein, a portion of theelastomeric block deflectable when the portion is actuated.

One embodiment of a method of microfabricating an elastomeric structurecomprises the steps of microfabricating a first elastomeric layer,microfabricating a second elastomeric layer; positioning the secondelastomeric layer on top of the first elastomeric layer, and bonding abottom surface of the second elastomeric layer onto a top surface of thefirst elastomeric layer.

A first alternative embodiment of a method of microfabricating anelastomeric structure comprises the steps of forming a first elastomericlayer on top of a first micromachined mold, the first micromachined moldhaving at least one first raised protrusion which forms at least onefirst channel in the bottom surface of the first elastomeric layer. Asecond elastomeric layer is formed on top of a second micromachinedmold, the second micromachined mold having at least one second raisedprotrusion which forms at least one second channel in the bottom surfaceof the second elastomeric layer. The bottom surface of the secondelastomeric layer is bonded onto a top surface of the first elastomericlayer such that the at least one second channel is enclosed between thefirst and second elastomeric layers.

A second alternative embodiment of a method of microfabricating anelastomeric structure in accordance with the present invention comprisesthe steps of forming a first elastomeric layer on top of a substrate,curing the first elastomeric layer, and depositing a first sacrificiallayer on the top surface of the first elastomeric layer. A portion ofthe first sacrificial layer is removed such that a first pattern ofsacrificial material remains on the top surface of the first elastomericlayer. A second elastomeric layer is formed over the first elastomericlayer thereby encapsulating the first pattern of sacrificial materialbetween the first and second elastomeric layers. The second elastomericlayer is cured and then sacrificial material is removed thereby formingat least one first recess between the first and second layers ofelastomer.

An embodiment of a method of actuating an elastomeric structure inaccordance with the present invention comprises providing an elastomericblock formed with first and second microfabricated recesses therein, thefirst and second microfabricated recesses being separated by a portionof the structure which is deflectable into either of the first or secondrecesses when the other of the first and second recesses. One of therecesses is pressurized such that the portion of the elastomericstructure separating the second recess from the first recess isdeflected into the other of the two recesses.

In other optional preferred aspects, magnetic or conductive materialscan be added to make layers of the elastomer magnetic or electricallyconducting, thus enabling the creation of all elastomer electromagneticdevices.

BRIEF DESCRIPTION OF THE DRAWINGS

Part I—FIGS. 1 to 7A illustrate successive steps of a first method offabricating the present invention, as follows:

FIG. 1 is an illustration of a first elastomeric layer formed on top ofa micromachined mold.

FIG. 2 is an illustration of a second elastomeric layer formed on top ofa micromachined mold.

FIG. 3 is an illustration of the elastomeric layer of FIG. 2 removedfrom the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1

FIG. 4 is an illustration corresponding to FIG. 3, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

FIG. 5 is an illustration corresponding to FIG. 4, but showing the firstand second elastomeric layers bonded together.

FIG. 6 is an illustration corresponding to FIG. 5, but showing the firstmicromachined mold removed and a planar substrate positioned in itsplace.

FIG. 7A is an illustration corresponding to FIG. 6, but showing theelastomeric structure sealed onto the planar substrate.

FIG. 7B is a front sectional view corresponding to FIG. 7A, showing anopen flow channel.

FIGS. 7C-7G are illustrations showing steps of a method for forming anelastomeric structure having a membrane formed from a separateelastomeric layer.

Part II—FIG. 7H show the closing of a first flow channel by pressurizinga second flow channel, as follows:

FIG. 7H corresponds to FIG. 7A, but shows a first flow channel closed bypressurization in second flow channel.

Part III—FIGS. 8 to 18 illustrate successive steps of a second method offabricating the present invention, as follows:

FIG. 8 is an illustration of a first elastomeric layer deposited on aplanar substrate.

FIG. 9 is an illustration showing a first photoresist layer deposited ontop of the first elastomeric layer of FIG. 8.

FIG. 10 is an illustration showing the system of FIG. 9, but with aportion of the first photoresist layer removed, leaving only a firstline of photoresist.

FIG. 11 is an illustration showing a second elastomeric layer applied ontop of the first elastomeric layer over the first line of photoresist ofFIG. 10, thereby encasing the photoresist between the first and secondelastomeric layers.

FIG. 12 corresponds to FIG. 11, but shows the integrated monolithicstructure produced after the first and second elastomer layers have beenbonded together.

FIG. 13 is an illustration showing a second photoresist layer depositedon top of the integral elastomeric structure of FIG. 12.

FIG. 14 is an illustration showing the system of FIG. 13, but with aportion of the second photoresist layer removed, leaving only a secondline of photoresist.

FIG. 15 is an illustration showing a third elastomer layer applied ontop of the second elastomeric layer and over the second line ofphotoresist of FIG. 14, thereby encapsulating the second line ofphotoresist between the elastomeric structure of FIG. 12 and the thirdelastomeric layer.

FIG. 16 corresponds to FIG. 15, but shows the third elastomeric layercured so as to be bonded to the monolithic structure composed of thepreviously bonded first and second elastomer layers.

FIG. 17 corresponds to FIG. 16, but shows the first and second lines ofphotoresist removed so as to provide two perpendicular overlapping, butnot intersecting, flow channels passing through the integratedelastomeric structure.

FIG. 18 is an illustration showing the system of FIG. 17, but with theplanar substrate thereunder removed.

Part IV—FIGS. 19 and 20 show further details of different flow channelcross-sections, as follows:

FIG. 19 shows a rectangular cross-section of a first flow channel.

FIG. 20 shows the flow channel cross section having a curved uppersurface.

Part V—FIGS. 21 to 24 show experimental results achieved by preferredembodiments of the present microfabricated valve:

FIG. 21 illustrates valve opening vs. applied pressure for various flowchannels.

FIG. 22 illustrates time response of a 100 μm×100 μm×10 μm RTVmicrovalve.

Part VI—FIGS. 23A to 33 show various microfabricated structures,networked together according to aspects of the present invention:

FIG. 23A is a top schematic view of an on/off valve.

FIG. 23B is a sectional elevation view along line 23B-23B in FIG. 23A

FIG. 24A is a top schematic view of a peristaltic pumping system.

FIG. 24B is a sectional elevation view along line 24B-24B in FIG. 24A

FIG. 25 is a graph showing experimentally achieved pumping rates vs.frequency for an embodiment of the peristaltic pumping system of FIG.24.

FIG. 26A is a top schematic view of one control line actuating multipleflow lines simultaneously.

FIG. 26B is a sectional elevation view along line 26B-26B in FIG. 26A

FIG. 27 is a schematic illustration of a multiplexed system adapted topermit flow through various channels.

FIG. 28A is a plan view of a flow layer of an addressable reactionchamber structure.

FIG. 28B is a bottom plan view of a control channel layer of anaddressable reaction chamber structure.

FIG. 28C is an exploded perspective view of the addressable reactionchamber structure formed by bonding the control channel layer of FIG.28B to the top of the flow layer of FIG. 28A.

FIG. 28D is a sectional elevation view corresponding to FIG. 28C, takenalong line 28D-28D in FIG. 28C.

FIG. 29 is a schematic of a system adapted to selectively direct fluidflow into any of an array of reaction wells.

FIG. 30 is a schematic of a system adapted for selectable lateral flowbetween parallel flow channels.

FIG. 31A is a bottom plan view of first layer (i.e.: the flow channellayer) of elastomer of a switchable flow array.

FIG. 31B is a bottom plan view of a control channel layer of aswitchable flow array.

FIG. 31C shows the alignment of the first layer of elastomer of FIG. 31Awith one set of control channels in the second layer of elastomer ofFIG. 31B.

FIG. 31D also shows the alignment of the first layer of elastomer ofFIG. 31A with the other set of control channels in the second layer ofelastomer of FIG. 31B.

FIG. 32 is a schematic of an integrated system for biopolymer synthesis.

FIG. 33 is a schematic of a further integrated system for biopolymersynthesis.

FIG. 34 is an optical micrograph of a section of a test structure havingseven layers of elastomer bonded together.

FIGS. 35A-35D show the steps of one embodiment of a method forfabricating an elastomer layer having a vertical via formed therein.

FIG. 36 shows one embodiment of a sorting apparatus in accordance withthe present invention.

FIG. 37 shows an embodiment of an apparatus for flowing process gasesover a semiconductor wafer in accordance with the present invention.

FIG. 38 shows an exploded view of one embodiment of a micro-mirror arraystructure in accordance with the present invention.

FIG. 39 shows a perspective view of a first embodiment of a refractivedevice in accordance with the present invention.

FIG. 40 shows a perspective view of a second embodiment of a refractivedevice in accordance with the present invention.

FIG. 41 shows a perspective view of a third embodiment of a refractivedevice in accordance with the present invention.

FIGS. 42A-42J show views of one embodiment of a normally-closed valvestructure in accordance with the present invention.

FIG. 43 shows a plan view of one embodiment of a device for performingseparations in accordance with the present invention.

FIGS. 44A-44D show plan views illustrating operation of one embodimentof a cell pen structure in accordance with the present invention.

FIGS. 45A-45B show plan and cross-sectional views illustrating operationof one embodiment of a cell cage structure in accordance with thepresent invention.

FIGS. 46A-46B show cross-sectional views illustrating operation of oneembodiment of a cell grinder structure in accordance with the presentinvention.

FIG. 47 shows a plan view of one embodiment of a pressure oscillatorstructure in accordance with the present invention.

FIGS. 48A and 48B show plan views illustrating operation of oneembodiment of a side-actuated valve structure in accordance with thepresent invention.

FIG. 49 plots Young's modulus versus percentage dilution of GE RTV 615elastomer with GE SF96-50 silicone fluid.

FIG. 50 shows a cross-sectional view of a structure in whichchannel-bearing faces are placed into contact to form a larger-sizedchannel.

FIG. 51 shows a cross-sectional view of a structure in which non-channelbearing faces are placed into contact and then sandwiched between twosubstrates.

FIGS. 52A-52C show cross-sectional views of the steps for constructing abridging structure. FIG. 52D shows a plan view of the bridgingstructure.

FIGS. 53A and 53 B show a cross-sectional view of one embodiment of acomposite structure in accordance with the present invention.

FIG. 54 shows a cross-sectional view of one embodiment of a compositestructure in accordance with the present invention.

FIGS. 55A-C show cross-sectional views of a process for formingelastomer structures by bonding along a vertical line.

FIG. 56 shows a schematic view of an electrolytically-actuated syringestructure in accordance with one embodiment of the present invention.

FIGS. 57A-57C illustrate cross-sectional views of a process for forminga flow channel having a membrane positioned therein.

FIGS. 58A-58D illustrate cross-sectional views of metering by volumeexclusion in accordance with an embodiment of the present invention.

FIGS. 59A-59B show cross-sectional and plan views respectively, of anembodiment of a linear amplifier constructed utilizing microfabricationtechniques in accordance with the present invention.

FIGS. 59C-59D show photographs of a linear amplifier in open and closedpositions, respectively.

FIG. 60 shows a plan view of an embodiment of a large multiplexerstructure in accordance with the present invention.

FIGS. 61A-61B show plan and cross-sectional views, respectively, of anembodiment of a one-way valve structure in accordance with the presentinvention.

FIGS. 62A-62E show cross-sectional views of steps of an embodiment of aprocess for forming a one-way valve in accordance with the presentinvention.

FIG. 63 is an embodiment of a NOR gate logic structure in accordancewith the present invention.

FIG. 64 is an embodiment of an AND gate logic structure in accordancewith the present invention which utilizes one way valve structures.

FIG. 65 plots light intensity versus cycle for an embodiment of a Braggmirror structure in accordance with the present invention.

FIG. 66 is a cross-sectional view of an embodiment of a tunablemicrolens structure in accordance with an embodiment of the presentinvention.

FIG. 67 is a plan view of a protein crystallization system in accordancewith one embodiment of the present invention.

FIG. 68A-68B are cross-sectional views of a method for bonding avertically-oriented microfabricated elastomer structure to ahorizontally-oriented microfabricated elastomer structure.

FIGS. 69A-B, illustrate a plan view of mixing steps performed by amicrofabricated structure in accordance another embodiment of thepresent invention.

FIG. 70 is a plan view of a protein crystallization system in accordancewith an alternative embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises a variety of microfabricated elastomericstructures which may be used as pumps or valves. Methods of fabricatingthe preferred elastomeric structures are also set forth.

Methods of Fabricating the Present Invention:

Two exemplary methods of fabricating the present invention are providedherein. It is to be understood that the present invention is not limitedto fabrication by one or the other of these methods. Rather, othersuitable methods of fabricating the present microstructures, includingmodifying the present methods, are also contemplated.

FIGS. 1 to 7B illustrate sequential steps of a first preferred method offabricating the present microstructure, (which may be used as a pump orvalve). FIGS. 8 to 18 illustrate sequential steps of a second preferredmethod of fabricating the present microstructure, (which also may beused as a pump or valve).

As will be explained, the preferred method of FIGS. 1 to 7B involvesusing pre-cured elastomer layers which are assembled and bonded.Conversely, the preferred method of FIGS. 8 to 18 involves curing eachlayer of elastomer “in place”. In the following description “channel”refers to a recess in the elastomeric structure which can contain a flowof fluid or gas.

The First Exemplary Method:

Referring to FIG. 1, a first micro-machined mold 10 is provided.Micro-machined mold 10 may be fabricated by a number of conventionalsilicon processing methods, including but not limited tophotolithography, ion-milling, and electron beam lithography.

As can be seen, micro-machined mold 10 has a raised line or protrusion11 extending therealong. A first elastomeric layer 20 is cast on top ofmold 10 such that a first recess 21 will be formed in the bottom surfaceof elastomeric layer 20, (recess 21 corresponding in dimension toprotrusion 11), as shown.

As can be seen in FIG. 2, a second micro-machined mold 12 having araised protrusion 13 extending therealong is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 will be formed in its bottom surface corresponding to thedimensions of protrusion 13.

As can be seen in the sequential steps illustrated in FIGS. 3 and 4,second elastomeric layer 22 is then removed from mold 12 and placed ontop of first elastomeric layer 20. As can be seen, recess 23 extendingalong the bottom surface of second elastomeric layer 22 will form a flowchannel 32.

Referring to FIG. 5, the separate first and second elastomeric layers 20and 22 (FIG. 4) are then bonded together to form an integrated (i.e.:monolithic) elastomeric structure 24.

As can been seen in the sequential step of FIGS. 6 and 7A, elastomericstructure 24 is then removed from mold 10 and positioned on top of aplanar substrate 14. As can be seen in FIGS. 7A and 7B, when elastomericstructure 24 has been sealed at its bottom surface to planar substrate14, recess 21 will form a flow channel 30.

The present elastomeric structures form a reversible hermetic seal withnearly any smooth planar substrate. An advantage to forming a seal thisway is that the elastomeric structures may be peeled up, washed, andre-used. In preferred aspects, planar substrate 14 is glass. A furtheradvantage of using glass is that glass is transparent, allowing opticalinterrogation of elastomer channels and reservoirs. Alternatively, theelastomeric structure may be bonded onto a flat elastomer layer by thesame method as described above, forming a permanent and high-strengthbond. This may prove advantageous when higher back pressures are used.

As can be seen in FIGS. 7A and 7B, flow channels 30 and 32 arepreferably disposed at an angle to one another with a small membrane 25of substrate 24 separating the top of flow channel 30 from the bottom offlow channel 32.

In preferred aspects, planar substrate 14 is glass. An advantage ofusing glass is that the present elastomeric structures may be peeled up,washed and reused. A further advantage of using glass is that opticalsensing may be employed. Alternatively, planar substrate 14 may be anelastomer itself, which may prove advantageous when higher backpressures are used.

The method of fabrication just described may be varied to form astructure having a membrane composed of an elastomeric materialdifferent than that forming the walls of the channels of the device.This variant fabrication method is illustrated in FIGS. 7C-7G.

Referring to FIG. 7C, a first micro-machined mold 10 is provided.Micro-machined mold 10 has a raised line or protrusion 11 extendingtherealong. In FIG. 7D, first elastomeric layer 20 is cast on top offirst micro-machined mold 10 such that the top of the first elastomericlayer 20 is flush with the top of raised line or protrusion 11. This maybe accomplished by carefully controlling the volume of elastomericmaterial spun onto mold 10 relative to the known height of raised line11. Alternatively, the desired shape could be formed by injectionmolding.

In FIG. 7E, second micro-machined mold 12 having a raised protrusion 13extending therealong is also provided. Second elastomeric layer 22 iscast on top of second mold 12 as shown, such that recess 23 is formed inits bottom surface corresponding to the dimensions of protrusion 13.

In FIG. 7F, second elastomeric layer 22 is removed from mold 12 andplaced on top of third elastomeric layer 222. Second elastomeric layer22 is bonded to third elastomeric layer 20 to form integral elastomericblock 224 using techniques described in detail below. At this point inthe process, recess 23 formerly occupied by raised line 13 will formflow channel 23.

In FIG. 7G, elastomeric block 224 is placed on top of firstmicro-machined mold 10 and first elastomeric layer 20. Elastomeric blockand first elastomeric layer 20 are then bonded together to form anintegrated (i.e.: monolithic) elastomeric structure 24 having a membranecomposed of a separate elastomeric layer 222.

When elastomeric structure 24 has been sealed at its bottom surface to aplanar substrate in the manner described above in connection with FIG.7A, the recess formerly occupied by raised line 11 will form flowchannel 30.

The variant fabrication method illustrated above in conjunction withFIGS. 7C-7G offers the advantage of permitting the membrane portion tobe composed of a separate material than the elastomeric material of theremainder of the structure. This is important because the thickness andelastic properties of the membrane play a key role in operation of thedevice. Moreover, this method allows the separate elastomer layer toreadily be subjected to conditioning prior to incorporation into theelastomer structure. As discussed in detail below, examples ofpotentially desirable condition include the introduction of magnetic orelectrically conducting species to permit actuation of the membrane,and/or the introduction of dopant into the membrane in order to alterits elasticity.

While the above method is illustrated in connection with forming variousshaped elastomeric layers formed by replication molding on top of amicromachined mold, the present invention is not limited to thistechnique. Other techniques could be employed to form the individuallayers of shaped elastomeric material that are to be bonded together.For example, a shaped layer of elastomeric material could be formed bylaser cutting or injection molding, or by methods utilizing chemicaletching and/or sacrificial materials as discussed below in conjunctionwith the second exemplary method.

The Second Exemplary Method:

A second exemplary method of fabricating an elastomeric structure whichmay be used as a pump or valve is set forth in the sequential stepsshown in FIGS. 8-18.

In this aspect of the invention, flow and control channels are definedby first patterning photoresist on the surface of an elastomeric layer(or other substrate, which may include glass) leaving a raised linephotoresist where a channel is desired. Next, a second layer ofelastomer is added thereover and a second photoresist is patterned onthe second layer of elastomer leaving a raised line photoresist where achannel is desired. A third layer of elastomer is deposited thereover.Finally, the photoresist is removed by dissolving it out of theelastomer with an appropriate solvent, with the voids formed by removalof the photoresist becoming the flow channels passing through thesubstrate.

Referring first to FIG. 8, a planar substrate 40 is provided. A firstelastomeric layer 42 is then deposited and cured on top of planarsubstrate 40. Referring to FIG. 9, a first photoresist layer 44A is thendeposited over the top of elastomeric layer 42. Referring to FIG. 10, aportion of photoresist layer 44A is removed such that only a first lineof photoresist 44B remains as shown. Referring to FIG. 11, a secondelastomeric layer 46 is then deposited over the top of first elastomericlayer 42 and over the first line of photoresist 44B as shown, therebyencasing first line of photoresist 44B between first elastomeric layer42 and second elastomeric layer 46. Referring to FIG. 12, elastomericlayers 46 is then cured on layer 42 to bond the layers together to forma monolithic elastomeric substrate 45.

Referring to FIG. 13, a second photoresist layer 48A is then depositedover elastomeric structure 45. Referring to FIG. 14, a portion of secondphotoresist layer 48A is removed, leaving only a second photoresist line48B on top of elastomeric structure 45 as shown. Referring to FIG. 15, athird elastomeric layer 50 is then deposited over the top of elastomericstructure 45 (comprised of second elastomeric layer 42 and first line ofphotoresist 44B) and second photoresist line 48B as shown, therebyencasing the second line of photoresist 48B between elastomericstructure 45 and third elastomeric layer 50.

Referring to FIG. 16, third elastomeric layer 50 and elastomericstructure 45 (comprising first elastomeric layer 42 and secondelastomeric layer 46 bonded together) is then bonded together forming amonolithic elastomeric structure 47 having photoresist lines 44B and 48Bpassing therethrough as shown. Referring to FIG. 17, photoresist lines44B, 48B are then removed (for example, by an solvent) such that a firstflow channel 60 and a second flow channel 62 are provided in theirplace, passing through elastomeric structure 47 as shown. Lastly,referring to FIG. 18, planar substrate 40 can be removed from the bottomof the integrated monolithic structure.

The method described in FIGS. 8-18 fabricates a patterned elastomerstructure utilizing development of photoresist encapsulated withinelastomer material. However, the methods in accordance with the presentinvention are not limited to utilizing photoresist. Other materials suchas metals could also serve as sacrificial materials to be removedselective to the surrounding elastomer material, and the method wouldremain within the scope of the present invention. For example, asdescribed in detail below in connection with FIGS. 35A-35D, gold metalmay be etched selective to RTV 615 elastomer utilizing the appropriatechemical mixture.

Preferred Layer and Channel Dimensions:

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels 30, 32, 60 and 62 preferably havewidth-to-depth ratios of about 10:1. A non-exclusive list of otherranges of width-to-depth ratios in accordance with embodiments of thepresent invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels 30, 32, 60 and 62 have widths of about 1 to 1000microns. A non-exclusive list of other ranges of widths of flow channelsin accordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels 30, 32, 60, and 62 have depths of about 1 to 100 microns.A non-exclusive list of other ranges of depths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250microns, and more preferably 1 to 100 microns, more preferably 2 to 20microns, and most preferably 5 to 10 microns. Exemplary channel depthsinclude including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm,and 250 μm.

The flow channels are not limited to these specific dimension ranges andexamples given above, and may vary in width in order to affect themagnitude of force required to deflect the membrane as discussed atlength below in conjunction with FIG. 27. For example, extremely narrowflow channels having a width on the order of 0.01 μm may be useful inoptical and other applications, as discussed in detail below.Elastomeric structures which include portions having channels of evengreater width than described above are also contemplated by the presentinvention, and examples of applications of utilizing such wider flowchannels include fluid reservoir and mixing channel structures.

Elastomeric layer 22 may be cast thick for mechanical stability. In anexemplary embodiment, layer 22 is 50 microns to several centimetersthick, and more preferably approximately 4 mm thick. A non-exclusivelist of ranges of thickness of the elastomer layer in accordance withother embodiments of the present invention is between about 0.1 micronto 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100 microns to 10 mm.

Accordingly, membrane 25 of FIG. 7B separating flow channels 30 and 32has a typical thickness of between about 0.01 and 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250, morepreferably 1 to 100 microns, more preferably 2 to 50 microns, and mostpreferably 5 to 40 microns. As such, the thickness of elastomeric layer22 is about 100 times the thickness of elastomeric layer 20. Exemplarymembrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm,0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm,15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm

Similarly, first elastomeric layer 42 may have a preferred thicknessabout equal to that of elastomeric layer 20 or 22; second elastomericlayer 46 may have a preferred thickness about equal to that ofelastomeric layer 20; and third elastomeric layer 50 may have apreferred thickness about equal to that of elastomeric layer 22.

Multilayer Soft Lithography Construction Techniques and Materials:

Soft Lithographic Bonding:

Preferably, elastomeric layers 20 and 22 (or elastomeric layers 42, 46and 50) are bonded together chemically, using chemistry that isintrinsic to the polymers comprising the patterned elastomer layers.Most preferably, the bonding comprises two component “addition cure”bonding.

In a preferred aspect, the various layers of elastomer are boundtogether in a heterogenous bonding in which the layers have a differentchemistry. Alternatively, a homogenous bonding may be used in which alllayers would be of the same chemistry. Thirdly, the respective elastomerlayers may optionally be glued together by an adhesive instead. In afourth aspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogenous aspect, the bonding process used tobind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

In an exemplary aspect of the present invention, elastomeric structuresare formed utilizing Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical.

In one embodiment in accordance with the present invention, two-layerelastomeric structures were fabricated from pure acrylated Urethane Ebe270. A thin bottom layer was spin coated at 8000 rpm for 15 seconds at170° C. The top and bottom layers were initially cured under ultravioletlight for 10 minutes under nitrogen utilizing a Model ELC 500 devicemanufactured by Electrolite corporation. The assembled layers were thencured for an additional 30 minutes. Reaction was catalyzed by a 0.5%vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals.The resulting elastomeric material exhibited moderate elasticity andadhesion to glass.

In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from a combination of25% Ebe 270/50% Irr245/25% isopropyl alcohol for a thin bottom layer,and pure acrylated Urethane Ebe 270 as a top layer. The thin bottomlayer was initially cured for 5 min, and the top layer initially curedfor 10 minutes, under ultraviolet light under nitrogen utilizing a ModelELC 500 device manufactured by Electrolite corporation. The assembledlayers were then cured for an additional 30 minutes. Reaction wascatalyzed by a 0.5% vol/vol mixture of Irgacure 500 manufactured byCiba-Geigy Chemicals. The resulting elastomeric material exhibitedmoderate elasticity and adhered to glass.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure as described above in FIGS. 8-18, bonding ofsuccessive elastomeric layers may be accomplished by pouring uncuredelastomer over a previously cured elastomeric layer and any sacrificialmaterial patterned thereupon. Bonding between elastomer layers occursdue to interpenetration and reaction of the polymer chains of an uncuredelastomer layer with the polymer chains of a cured elastomer layer.Subsequent curing of the elastomeric layer will create a bond betweenthe elastomeric layers and create a monolithic elastomeric structure.

Referring to the first method of FIGS. 1 to 7B, first elastomeric layer20 may be created by spin-coating an RTV mixture on microfabricated mold12 at 2000 rpm's for 30 seconds yielding a thickness of approximately 40microns. Second elastomeric layer 22 may be created by spin-coating anRTV mixture on microfabricated mold 11. Both layers 20 and 22 may beseparately baked or cured at about 80° C. for 1.5 hours. The secondelastomeric layer 22 may be bonded onto first elastomeric layer 20 atabout 80° C. for about 1.5 hours.

Micromachined molds 10 and 12 may be patterned photoresist on siliconwafers. In an exemplary aspect, a Shipley SIR 5740 photoresist was spunat 2000 rpm patterned with a high resolution transparency film as a maskand then developed yielding an inverse channel of approximately 10microns in height. When baked at approximately 200° C. for about 30minutes, the photoresist reflows and the inverse channels becomerounded. In preferred aspects, the molds may be treated withtrimethylchlorosilane (TMCS) vapor for about a minute before each use inorder to prevent adhesion of silicone rubber.

Using the various multilayer soft lithography construction techniquesand materials set forth herein, the present inventors haveexperimentally succeeded in creating channel networks comprises of up toseven separate elastomeric layers thick, with each layer being about 40μm thick. It is foreseeable that devices comprising more than sevenseparate elastomeric layers bonded together could be developed.

While the above discussion focuses upon methods for bonding togethersuccessive horizontal layers of elastomer, in alternative embodiments inaccordance with the present invention bonding could also occur betweenvertical interfaces of different portions of an elastomer structure. Oneembodiment of such an alternative approach is shown in FIGS. 55A-55C,which illustrate cross-sectional views of a process for forming such adevice utilizing separation of an elastomer structure along a flowchannel section.

FIG. 55A shows a cross-section of two devices 6000 and 6010 comprisingrespective first elastomer layers 6002 and 6012 overlying secondelastomer layers 6004 and 6014, thereby defining respective flowchannels 6006 and 6016. First elastomer layer 6002 comprises a firstelastomer material, and second elastomer layer 6004 comprises a secondelastomer material configured to bond with the first elastomer material.Conversely, first elastomer layer 6012 comprises the second elastomermaterial and second elastomer layer 6014 comprises the first elastomermaterial.

FIG. 55B shows the results of cutting devices 6000 and 6010 alongvertical lines 6001 and 6011 extending along the length of flow channels6006 and 6016, respectively, to form half structures 6000 a-b and 6010a-b.

FIG. 55C shows the results of cross-combining the half structures, suchthat first half 6000 a is bonded to second half 6010 b to form device6008, and first half 6010 a is bonded to second half 6000 b to formdevice 6018. This reassembly is enabled by the bonding between the firstelastomer material and the second elastomer material.

The bonding method just described can be useful for the assembly ofmultiple chips together, edge to edge, to form a multi-chip module. Thisis particularly advantageous for the creation of microfluidic modules,each having a particular function, which can be assembled from componentchips as desired.

While the above description focuses upon bonding of successive layersfeaturing horizontally-oriented channels, it is also possible tofabricate a layer having a horizontally-oriented channel, and then toorient the layer in a vertical position and bond the vertically-orientedlayer to a horizontally oriented layer. This is shown in FIG. 68A-B,which illustrates cross-sectional views of a method for bonding avertically-oriented microfabricated elastomer structure to ahorizontally-oriented microfabricated elastomer structure.

FIG. 68A shows first horizontally-oriented microfabricated elastomerstructure 7300 comprising control channel 7302 formed in first elastomerlayer 7304 and overlying and separated from flow channel 7306 bymembrane 7308 formed from second elastomer layer 7310.

Second elastomer layer 7310 also defines a vertical via 7312 incommunication with flow channel 7306, and first elastomer layer 7304does not extend completely over the top of second elastomer layer 7310.Second horizontally-oriented microfabricated elastomer structure 7320similarly comprises control channel 7322 formed in third elastomer layer7324 and overlying and separated from flow channel 7326 by membrane 7328formed from fourth elastomer layer 7330.

FIG. 68B shows assembly of a compound microfabricated elastomericstructure 7340 by orienting second microfabricated structure 7320vertically on end, and placing second structure 7320 into contact withfirst microfabricated structure 7300 such that flow channel 7326 incontact with via 7312. By employing compound structure 7340, fluids canbe withdrawn from or introduced into flow channel 7306 through via 7312.

Suitable Elastomeric Materials:

Allcock et al, Contemporary Polymer Chemistry, 2^(nd) Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa-1TPa, more preferably between about 10 Pa-100 GPa, more preferablybetween about 20 Pa-1 GPa, more preferably between about 50 Pa-10 MPa,and more preferably between about 100 Pa-1 MPa are useful in accordancewith the present invention, although elastomeric materials having aYoung's modulus outside of these ranges could also be utilized dependingupon the needs of a particular application.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, elastomeric layers 20,22, 42, 46 and 50 may preferably be fabricated from silicone rubber.However, other suitable elastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones.

Polyisoprene, Polybutadiene, Polychloroprene:

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

Polyisobutylene:

Pure polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (˜1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thepolyisobutylene backbone, which may then be vulcanized as above.

Poly(styrene-butadiene-styrene):

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

Polyurethanes:

Polyurethanes are produced from di-isocyanates (A-A) and di-alcohols ordi-amines (B-B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, would make themuseful for heterogeneous multilayer soft lithography just as RTV 615 is:by using excess A-A in one layer and excess B-B in the other layer.

Silicones:

Silicone polymers probably have the greatest structural variety, andalmost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

Cross Linking Agents:

In addition to the use of the simple “pure” polymers discussed above,crosslinking agents may be added. Some agents (like the monomers bearingpendant double bonds for vulcanization) are suitable for allowinghomogeneous (A to A) multilayer soft lithography or photoresistencapsulation; in such an approach the same agent is incorporated intoboth elastomer layers. Complementary agents (i.e. one monomer bearing apendant double bond, and another bearing a pendant Si—H group) aresuitable for heterogeneous (A to B) multilayer soft lithography. In thisapproach complementary agents are added to adjacent layers.

Other Materials:

In addition, polymers incorporating materials such as chlorosilanes ormethyl-, ethyl-, and phenylsilanes, and polydimethylsiloxane (PDMS) suchas Dow Chemical Corp. Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical may also be used.

The following is a non-exclusive list of elastomeric materials which maybe utilized in connection with the present invention: polyisoprene,polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and siliconepolymers; or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F),poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene)(nitrile rubber), poly(l-butene),poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F),poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidenefluoride—hexafluoropropylene) copolymer (Viton), elastomericcompositions of polyvinylchloride (PVC), polysulfone, polycarbonate,polymethylmethacrylate (PMMA), and polytertrafluoroethylene (Teflon).

Doping and Dilution:

Elastomers may also be “doped” with uncrosslinkable polymer chains ofthe same class. For instance RTV 615 may be diluted with GE SF96-50Silicone Fluid. This serves to reduce the viscosity of the uncuredelastomer and reduces the Young's modulus of the cured elastomer.Essentially, the crosslink-capable polymer chains are spread furtherapart by the addition of “inert” polymer chains, so this is called“dilution”. RTV 615 cures at up to 90% dilution, with a dramaticreduction in Young's modulus.

FIG. 49 plots Young's modulus versus percentage dilution with GE SF96-50diluent of GE RTV 615 elastomer having a ratio of 30:1 A:B. FIG. 49shows that the flexibility of the elastomer material, and hence theresponsiveness of the valve membrane to an applied actuation force, canbe controlled during fabrication of the device.

Other examples of doping of elastomer material may include theintroduction of electrically conducting or magnetic species, asdescribed in detail below in conjunction with alternative methods ofactuating the membrane of the device. Should it be desired, doping withfine particles of material having an index of refraction different thanthe elastomeric material (i.e. silica, diamond, sapphire) is alsocontemplated as a system for altering the refractive index of thematerial. Strongly absorbing or opaque particles may be added to renderthe elastomer colored or opaque to incident radiation. This mayconceivably be beneficial in an optically addressable system.

Finally, by doping the elastomer with specific chemical species, thesedoped chemical species may be presented at the elastomer surface, thusserving as anchors or starting points for further chemicalderivitization.

Pre-Treatment and Surface Coating

Once the elastomeric material has been molded or etched into theappropriate shape, it may be necessary to pre-treat the material inorder to facilitate operation in connection with a particularapplication.

For example, one possible application for an elastomeric device inaccordance with the present invention is to sort biological entitiessuch as cells or DNA. In such an application, the hydrophobic nature ofthe biological entity may cause it to adhere to the hydrophobicelastomer of the walls of the channel. Therefore, it may be useful topre-treat the elastomeric structure order to impart a hydrophiliccharacter to the channel walls. In an embodiment of the presentinvention utilizing the General Electric RTV 615 elastomer, this can beaccomplished by boiling the shaped elastomer in acid (e.g. 0.01% HCl inwater, pH 2.7, at 60° C. for 40 min).

Other types of pre-treatment of elastomer material are also contemplatedby the present application. For example, certain portions of elastomermay be pre-treated to create anchors for surface chemistry reactions(for example in the formation of peptide chains), or binding sites forantibodies, as would be advantageous in a given application. Otherexamples of pre-treatment of elastomer material may include theintroduction of reflective material on the elastomer surface, asdescribed in detail below in conjunction with the micro-mirror arrayapplication.

Methods of Operating the Present Invention:

FIGS. 7B and 7H together show the closing of a first flow channel bypressurizing a second flow channel, with FIG. 7B (a front sectional viewcutting through flow channel 32 in corresponding FIG. 7A), showing anopen first flow channel 30; with FIG. 7H showing first flow channel 30closed by pressurization of the second flow channel 32.

Referring to FIG. 7B, first flow channel 30 and second flow channel 32are shown.

Membrane 25 separates the flow channels, forming the top of first flowchannel 30 and the bottom of second flow channel 32. As can be seen,flow channel 30 is “open”.

As can be seen in FIG. 7H, pressurization of flow channel 32 (either bygas or liquid introduced therein) causes membrane 25 to deflectdownward, thereby pinching off flow F passing through flow channel 30.Accordingly, by varying the pressure in channel 32, a linearly actuablevalving system is provided such that flow channel 30 can be opened orclosed by moving membrane 25 as desired. (For illustration purposesonly, channel 30 in FIG. 7G is shown in a “mostly closed” position,rather than a “fully closed” position).

It is to be understood that exactly the same valve opening and closingmethods can be achieved with flow channels 60 and 62.

Since such valves are actuated by moving the roof of the channelsthemselves (i.e.: moving membrane 25) valves and pumps produced by thistechnique have a truly zero dead volume, and switching valves made bythis technique have a dead volume approximately equal to the activevolume of the valve, for example about 100×100×10 μm=100 μL. Such deadvolumes and areas consumed by the moving membrane are approximately twoorders of magnitude smaller than known conventional microvalves. Smallerand larger valves and switching valves are contemplated in the presentinvention, and a non-exclusive list of ranges of dead volume includes 1aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to100 pL

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.10 The smallest known volumes capableof being metered by automated systems is about ten-times larger (1 μl).Utilizing pumps and valves in accordance with the present invention,volumes of liquid of 10 nl or smaller can routinely be metered anddispensed. The accurate metering of extremely small volumes of fluidenabled by the present invention would be extremely valuable in a largenumber of biological applications, including diagnostic tests andassays.

Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:

w=(BPb ⁴)/(Eh ³),  (1)

where:

-   -   w=deflection of plate;    -   B=shape coefficient (dependent upon length vs. width and support        of edges of plate);    -   P=applied pressure;    -   b=plate width    -   E=Young's modulus; and        h=plate thickness.

Thus even in this extremely simplified expression, deflection of anelastomeric membrane in response to a pressure will be a function of:the length, width, and thickness of the membrane, the flexibility of themembrane (Young's modulus), and the applied actuation force.

Because each of these parameters will vary widely depending upon theactual dimensions and physical composition of a particular elastomericdevice in accordance with the present invention, a wide range ofmembrane thicknesses and elasticities, channel widths, and actuationforces are contemplated by the present invention.

It should be understood that the formula just presented is only anapproximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

FIGS. 21a and 21b illustrate valve opening vs. applied pressure for a100 μm wide first flow channel 30 and a 50 μm wide second flow channel32. The membrane of this device was formed by a layer of GeneralElectric Silicones RTV 615 having a thickness of approximately 30 μm anda Young's modulus of approximately 750 kPa. FIGS. 21a and 21b show theextent of opening of the valve to be substantially linear over most ofthe range of applied pressures.

Air pressure was applied to actuate the membrane of the device through a10 cm long piece of plastic tubing having an outer diameter of 0.025″connected to a 25 mm piece of stainless steel hypodermic tubing with anouter diameter of 0.025″ and an inner diameter of 0.013″. This tubingwas placed into contact with the control channel by insertion into theelastomeric block in a direction normal to the control channel. Airpressure was applied to the hypodermic tubing from an external LHDAminiature solenoid valve manufactured by Lee Co.

Connection of conventional microfluidic devices to an external fluidflow poses a number of problems avoided by the external configurationjust described. One such problem is the fragility of their connectionswith the external environment. Specifically, conventional microfluidicdevices are composed of hard, inflexible materials (such as silicon), towhich pipes or tubing allowing connection to external elements must bejoined. The rigidity of the conventional material creates significantphysical stress at points of contact with small and delicate externaltubing, rendering conventional microfluidic devices prone to fractureand leakage at these contact points.

By contrast, the elastomer of the present invention is flexible and canbe easily penetrated for external connection by a tube composed a hardmaterial. For example, in an elastomer structure fabricated utilizingthe method shown in FIGS. 1-7B, a hole extending from the exteriorsurface of the structure into the control channel can be made bypenetrating the elastomer with metal hypodermic tubing after the upperelastomer piece has been removed from the mold (as shown in FIG. 3) andbefore this piece has been bonded to the lower elastomer piece (as shownin FIG. 4). Between these steps, the roof of the control channel isexposed to the user's view and is accessible to insertion and properpositioning of the hole. Following completion of fabrication of thedevice, the metal hypodermic tubing is inserted into the hole tocomplete the fluid connection.

Moreover, the elastomer of the present invention will flex in responseto physical strain at the point of contact with an external connection,rendering the external physical connection more robust. This flexibilitysubstantially reduces the chance of leakage or fracture of the presentdevice.

Another disadvantage of conventional microfluidic devices is thedifficulty in establishing an effective seal between the device and itsexternal links. Because of the extremely narrow diameter of the channelsof these devices, even moderate rates of fluid flow can requireextremely high pressures. Unwanted leakage at the junction between thedevice and external connections may result. However, the flexibility ofthe elastomer of the present device also aids in overcoming leakagerelating to pressure. In particular, the flexible elastomeric materialflexes to conform around inserted tubing in order to form a pressureresistant seal.

While control of the flow of material through the device has so far beendescribed utilizing applied gas pressure, other fluids could be used.

For example, air is compressible, and thus experiences some finite delaybetween the time of application of pressure by the external solenoidvalve and the time that this pressure is experienced by the membrane. Inan alternative embodiment of the present invention, pressure could beapplied from an external source to a noncompressible fluid such as wateror hydraulic oils, resulting in a near-instantaneous transfer of appliedpressure to the membrane. However, if the displaced volume of the valveis large or the control channel is narrow, higher viscosity of a controlfluid may contribute to delay in actuation. The optimal medium fortransferring pressure will therefore depend upon the particularapplication and device configuration, and both gaseous and liquid mediaare contemplated by the invention.

While external applied pressure as described above has been applied by apump/tank system through a pressure regulator and external miniaturevalve, other methods of applying external pressure are also contemplatedin the present invention, including gas tanks, compressors, pistonsystems, and columns of liquid. Also contemplated is the use ofnaturally occurring pressure sources such as may be found inside livingorganisms, such as blood pressure, gastric pressure, the pressurepresent in the cerebro-spinal fluid, pressure present in theintra-ocular space, and the pressure exerted by muscles during normalflexure. Other methods of regulating external pressure are alsocontemplated, such as miniature valves, pumps, macroscopic peristalticpumps, pinch valves, and other types of fluid regulating equipment suchas is known in the art.

As can be seen, the response of valves in accordance with embodiments ofthe present invention have been experimentally shown to be almostperfectly linear over a large portion of its range of travel, withminimal hysteresis. Accordingly, the present valves are ideally suitedfor microfluidic metering and fluid control. The linearity of the valveresponse demonstrates that the individual valves are well modeled asHooke's Law springs. Furthermore, high pressures in the flow channel(i.e.: back pressure) can be countered simply by increasing theactuation pressure. Experimentally, the present inventors have achievedvalve closure at back pressures of 70 kPa, but higher pressures are alsocontemplated. The following is a nonexclusive list of pressure rangesencompassed by the present invention: 10 Pa-25 MPa; 100 Pa-10 Mpa, 1kPa-1 MPa, 1 kPa-300 kPa, 5 kPa-200 kPa, and 15 kPa-100 kPa.

While valves and pumps do not require linear actuation to open andclose, linear response does allow valves to more easily be used asmetering devices. In one embodiment of the invention, the opening of thevalve is used to control flow rate by being partially actuated to aknown degree of closure. Linear valve actuation makes it easier todetermine the amount of actuation force required to close the valve to adesired degree of closure. Another benefit of linear actuation is thatthe force required for valve actuation may be easily determined from thepressure in the flow channel. If actuation is linear, increased pressurein the flow channel may be countered by adding the same pressure (forceper unit area) to the actuated portion of the valve.

Linearity of a valve depends on the structure, composition, and methodof actuation of the valve structure. Furthermore, whether linearity is adesirable characteristic in a valve depends on the application.Therefore, both linearly and non-linearly actuatable valves arecontemplated in the present invention, and the pressure ranges overwhich a valve is linearly actuatable will vary with the specificembodiment.

FIG. 22 illustrates time response (i.e.: closure of valve as a functionof time in response to a change in applied pressure) of a 100 μm×100μm×10 μm RTV microvalve with 10-cm-long air tubing connected from thechip to a pneumatic valve as described above.

Two periods of digital control signal, actual air pressure at the end ofthe tubing and valve opening are shown in FIG. 22. The pressure appliedon the control line is 100 kPa, which is substantially higher than the˜40 kPa required to close the valve. Thus, when closing, the valve ispushed closed with a pressure 60 kPa greater than required. Whenopening, however, the valve is driven back to its rest position only byits own spring force (≦40 kPa). Thus, τ_(close) is expected to besmaller than τ_(open). There is also a lag between the control signaland control pressure response, due to the limitations of the miniaturevalve used to control the pressure. Calling such lags t and the 1/e timeconstants τ, the values are: t_(open), =3.63 ms, τ_(open), =1.88 ms,t_(close)=2.15 ms, τ_(close), =0.51 ms. If 3τ each are allowed foropening and closing, the valve runs comfortably at 75 Hz when filledwith aqueous solution.

If one used another actuation method which did not suffer from openingand closing lag, this valve would run at ˜375 Hz. Note also that thespring constant can be adjusted by changing the membrane thickness; thisallows optimization for either fast opening or fast closing. The springconstant could also be adjusted by changing the elasticity (Young'smodulus) of the membrane, as is possible by introducing dopant into themembrane or by utilizing a different elastomeric material to serve asthe membrane (described above in conjunction with FIGS. 7C-7H.)

When experimentally measuring the valve properties as illustrated inFIGS. 21 and 22, the valve opening was measured by fluorescence. Inthese experiments, the flow channel was filled with a solution offluorescein isothiocyanate (FITC) in buffer (pH 8) and the fluorescenceof a square area occupying the center ˜⅓rd of the channel is monitoredon an epi-fluorescence microscope with a photomultiplier tube with a 10kHz bandwidth. The pressure was monitored with a Wheatstone-bridgepressure sensor (SenSym SCC15GD2) pressurized simultaneously with thecontrol line through nearly identical pneumatic connections.

Flow Channel Cross Sections:

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of the lower flow channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper cross channel). Such a curved upper surface facilitates valvesealing, as follows.

Referring to FIG. 19, a cross sectional view (similar to that of FIG.7B) through flow channels 30 and 32 is shown. As can be seen, flowchannel 30 is rectangular in cross sectional shape. In an alternatepreferred aspect of the invention, as shown in FIG. 20, thecross-section of a flow channel 30 instead has an upper curved surface.

Referring first to FIG. 19, when flow channel 32 is pressurized, themembrane portion 25 of elastomeric block 24 separating flow channels 30and 32 will move downwardly to the successive positions shown by thedotted lines 25A, 25B, 25C, 25D, and 25E. As can be seen, incompletesealing may possibly result at the edges of flow channel 30 adjacentplanar substrate 14.

In the alternate preferred embodiment of FIG. 20, flow channel 30 a hasa curved upper wall 25A. When flow channel 32 is pressurized, membraneportion 25 will move downwardly to the successive positions shown bydotted lines 25A2, 25A3, 25A4 and 25A5, with edge portions of themembrane moving first into the flow channel, followed by top membraneportions. An advantage of having such a curved upper surface at membrane25A is that a more complete seal will be provided when flow channel 32is pressurized. Specifically, the upper wall of the flow channel 30 willprovide a continuous contacting edge against planar substrate 14,thereby avoiding the “island” of contact seen between wall 25 and thebottom of flow channel 30 in FIG. 19.

Another advantage of having a curved upper flow channel surface atmembrane 25A is that the membrane can more readily conform to the shapeand volume of the flow channel in response to actuation. Specifically,where a rectangular flow channel is employed, the entire perimeter (2×flow channel height, plus the flow channel width) must be forced intothe flow channel. However where an arched flow channel is used, asmaller perimeter of material (only the semi-circular arched portion)must be forced into the channel. In this manner, the membrane requiresless change in perimeter for actuation and is therefore more responsiveto an applied actuation force to block the flow channel

In an alternate aspect, (not illustrated), the bottom of flow channel 30is rounded such that its curved surface mates with the curved upper wall25A as seen in FIG. 20 described above.

In summary, the actual conformational change experienced by the membraneupon actuation will depend upon the configuration of the particularelastomeric structure. Specifically, the conformational change willdepend upon the length, width, and thickness profile of the membrane,its attachment to the remainder of the structure, and the height, width,and shape of the flow and control channels and the material propertiesof the elastomer used. The conformational change may also depend uponthe method of actuation, as actuation of the membrane in response to anapplied pressure will vary somewhat from actuation in response to amagnetic or electrostatic force.

Moreover, the desired conformational change in the membrane will alsovary depending upon the particular application for the elastomericstructure. In the simplest embodiments described above, the valve mayeither be open or closed, with metering to control the degree of closureof the valve. In other embodiments however, it may be desirable to alterthe shape of the membrane and/or the flow channel in order to achievemore complex flow regulation. For instance, the flow channel could beprovided with raised protrusions beneath the membrane portion, such thatupon actuation the membrane shuts off only a percentage of the flowthrough the flow channel, with the percentage of flow blockedinsensitive to the applied actuation force.

Many membrane thickness profiles and flow channel cross-sections arecontemplated by the present invention, including rectangular,trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, andpolygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as the embodiment with protrusionsdiscussed immediately above or an embodiment having concavities in theflow channel, are also contemplated by the present invention.

In addition, while the invention is described primarily above inconjunction with an embodiment wherein the walls and ceiling of the flowchannel are formed from elastomer, and the floor of the channel isformed from an underlying substrate, the present invention is notlimited to this particular orientation. Walls and floors of channelscould also be formed in the underlying substrate, with only the ceilingof the flow channel constructed from elastomer. This elastomer flowchannel ceiling would project downward into the channel in response toan applied actuation force, thereby controlling the flow of materialthrough the flow channel. In general, monolithic elastomer structures asdescribed elsewhere in the instant application are preferred formicrofluidic applications. However, it may be useful to employ channelsformed in the substrate where such an arrangement provides advantages.For instance, a substrate including optical waveguides could beconstructed so that the optical waveguides direct light specifically tothe side of a microfluidic channel.

Alternate Valve Actuation Techniques:

In addition to pressure based actuation systems described above,optional electrostatic and magnetic actuation systems are alsocontemplated, as follows.

Electrostatic actuation can be accomplished by forming oppositelycharged electrodes (which will tend to attract one another when avoltage differential is applied to them) directly into the monolithicelastomeric structure. For example, referring to FIG. 7B, an optionalfirst electrode 70 (shown in phantom) can be positioned on (or in)membrane 25 and an optional second electrode 72 (also shown in phantom)can be positioned on (or in) planar substrate 14. When electrodes 70 and72 are charged with opposite polarities, an attractive force between thetwo electrodes will cause membrane 25 to deflect downwardly, therebyclosing the “valve” (i.e.: closing flow channel 30).

For the membrane electrode to be sufficiently conductive to supportelectrostatic actuation, but not so mechanically stiff so as to impedethe valve's motion, a sufficiently flexible electrode must be providedin or over membrane 25. Such an electrode may be provided by a thinmetallization layer, doping the polymer with conductive material, ormaking the surface layer out of a conductive material.

In an exemplary aspect, the electrode present at the deflecting membranecan be provided by a thin metallization layer which can be provided, forexample, by sputtering a thin layer of metal such as 20 nm of gold. Inaddition to the formation of a metallized membrane by sputtering, othermetallization approaches such as chemical epitaxy, evaporation,electroplating, and electroless plating are also available. Physicaltransfer of a metal layer to the surface of the elastomer is alsoavailable, for example by evaporating a metal onto a flat substrate towhich it adheres poorly, and then placing the elastomer onto the metaland peeling the metal off of the substrate.

A conductive electrode 70 may also be formed by depositing carbon black(i.e. Cabot Vulcan XC72R) on the elastomer surface, either by wiping onthe dry powder or by exposing the elastomer to a suspension of carbonblack in a solvent which causes swelling of the elastomer, (such as achlorinated solvent in the case of PDMS). Alternatively, the electrode70 may be formed by constructing the entire layer 20 out of elastomerdoped with conductive material (i.e. carbon black or finely dividedmetal particles). Yet further alternatively, the electrode may be formedby electrostatic deposition, or by a chemical reaction that producescarbon. In experiments conducted by the present inventors, conductivitywas shown to increase with carbon black concentration from 5.6×10⁻¹⁶ toabout 5×10⁻³ (Ω-cm)⁻¹. The lower electrode 72, which is not required tomove, may be either a compliant electrode as described above, or aconventional electrode such as evaporated gold, a metal plate, or adoped semiconductor electrode.

A pump or valve structure in accordance with embodiments of the presentinvention could also be actuated by applying electrical potential tomaterials whose physical properties change in an electro-magnetic field.One example of such a material is liquid mercury, whose surface tensionchanges in an applied electromagnetic field. In this method ofactuation, a pocket of liquid mercury is present in the control channeloverlying the flow channel. The mercury filled pocket is connected toelectrodes. In response to the voltage applied across the electrode,liquid mercury within the control experiences a change in surfacetension, changing the adjacent membrane from a relaxed state to anon-relaxed state. Depending upon the particular structure of the valve,this changed state may correspond to either opening or closing thevalve. Upon cessation of the applied voltage, mercury within the controlchannel will resume its original surface tension, and the membrane willassume is relaxed position.

In a similar approach, surfaces of a control channel may be coated witha material that changes shape in response to an applied potential. Forexample, polypyrrole is a conjugated polymer which changes shape withinan electrolyte bearing an applied voltage. A control channel may becoated with polypyrrole or another organic conductor which changesmacroscopic structure in response to an applied electric field. Thecoated control channel is then filled with electrolyte, and theelectrolyte placed into contact with electrodes. When a potentialdifference is applied across the electrolyte, the coated channel isattracted to the electrode. Thus by designing the proper placement ofthe polypyrrole coated channel in relation to the electrode, the flowchannel can be selectively opened or closed by applying a current.

Magnetic actuation of the flow channels can be achieved by fabricatingthe membrane separating the flow channels with a magneticallypolarizable material such as iron, or a permanently magnetized materialsuch as polarized NdFeB. In experiments conducted by the presentinventors, magnetic silicone was created by the addition of iron powder(about 1 um particle size), up to 20% iron by weight.

Where the membrane is fabricated with a magnetically polarizablematerial, the membrane can be actuated by attraction in response to anapplied magnetic field Where the membrane is fabricated with a materialcapable of maintaining permanent magnetization, the material can firstbe magnetized by exposure to a sufficiently high magnetic field, andthen actuated either by attraction or repulsion in response to thepolarity of an applied inhomogenous magnetic field.

The magnetic field causing actuation of the membrane can be generated ina variety of ways. In one embodiment, the magnetic field is generated byan extremely small inductive coil formed in or proximate to theelastomer membrane. The actuation effect of such a magnetic coil wouldbe localized, allowing actuation of individual pump and/or valvestructures. Alternatively, the magnetic field could be generated by alarger, more powerful source, in which case actuation would be globaland would actuate multiple pump and/or valve structures at one time.

It is further possible to combine pressure actuation with electrostaticor magnetic actuation. Specifically, a bellows structure in fluidcommunication with a recess could be electrostatically or magneticallyactuated to change the pressure in the recess and thereby actuate amembrane structure adjacent to the recess.

In addition to electrical or magnetic actuation as described above,optional electrolytic and electrokinetic actuation systems are alsocontemplated by the present invention.

For example, actuation pressure on the membrane could arise from anelectrolytic reaction in a recess (control channel) overlying themembrane. In such an embodiment, electrodes present in the recess wouldapply a voltage across an electrolyte in the recess. This potentialdifference would cause electrochemical reaction at the electrodes andresult in the generation of gas, in turn giving rise to a pressuredifferential in the recess. Depending upon the elastomer type and theexact device structure, gas generated by such an electrolytic reactioncould be mechanically vented or allowed to slowly diffuse out of thepolymer matrix of the elastomer, releasing the pressure generated withinthe pocket and returning the membrane to its relaxed state.

Alternatively, rather than relying on outdiffusion for reversibility ofactuation, the gases could be recombined to form water, releasingpressure and returning the membrane to its relaxed state. Formation ofwater from hydrogen and water vapor can be accomplished through the useof a catalyst, or by providing sufficient energy to cause controlledcombustion.

One potential application of an electrolytic method of actuation is themicrosyringe structure shown in FIG. 56. FIG. 56 is a schematic view ofan elastomer block bearing a flow channel 6100 in fluid communicationwith first, second, and third chambers 6102, 6104, and 6106respectively. First chamber 6102 contains a salt solution 6103 incontact with electrodes 6108 and 6110. Third chamber 6106 contains drug6107 or other liquid material that is to be output from the fluidicdevice through output channel 6112. Second chamber 6104 contains aninert oil 6105 intended to physically isolate drug 6107 from saltsolution 6103. Upon application of a potential difference acrosselectrodes 6108 and 6110, salt solution 6103 undergoes electrolysis,generating gases such that first chamber 6102 rapidly experiences anelevation in pressure. This elevation in pressure causes oil 6105 toflow from second chamber 6104 into third chamber 6106, displacing drug6107 and causing the flow of drug 6107 from third chamber 6106 to outputchannel 6112 and ultimately into a patient.

An embodiment of a method for actuating a microfabricated elastomerstructure comprises providing an aqueous salt solution in a controlrecess formed in an elastomeric block and overlying and separated from aflow channel by an elastomer membrane, applying a potential differenceto the salt solution to generate a gas, such that a pressure in thecontrol recess causes the membrane to deflect into the flow channel.

An embodiment of a microfabricated syringe structure in accordance withthe present invention comprises a first chamber formed in an elastomericblock and including an aqueous salt solution, a first electrode, and asecond electrode. A second chamber is formed in the elastomeric blockand contains an inert liquid, the second chamber in fluid communicationwith the first chamber through a first flow channel. A third chamber isformed in the elastomeric block and containing an ejectable material,the third chamber in fluid communication with the second chamber througha second flow channel and in fluid communication with an environmentthrough an outlet, such that application of a potential differenceacross the electrodes generates gas in the first chamber, the gasdisplacing the inert material into the third chamber, the inert materialdisplacing the injectable material into the environment.

Actuation pressure on the membrane of a device in accordance withembodiments of the present invention could also arise from anelectrokinetic fluid flow in the control channel. In such an embodiment,electrodes present at opposite ends of the control channel would apply apotential difference across an electrolyte present in the controlchannel. Electrokinetic flow induced by the potential difference couldgive rise to a pressure differential.

Finally, it is also possible to actuate the device by causing a fluidflow in the control channel based upon the application of thermalenergy, either by thermal expansion or by production of gas from liquid.For example, in one alternative embodiment in accordance with thepresent invention, a pocket of fluid (e.g. in a fluid-filled controlchannel) is positioned over the flow channel. Fluid in the pocket can bein communication with a temperature variation system, for example aheater. Thermal expansion of the fluid, or conversion of material fromthe liquid to the gas phase, could result in an increase in pressure,closing the adjacent flow channel. Subsequent cooling of the fluid wouldrelieve pressure and permit the flow channel to open.

Alternatively, or in conjunction with simple cooling, heated vapor fromthe fluid pocket could diffuse out of the elastomer material, therebyrelieving pressure and permitting the flow channel to open. Fluid lostfrom the control channel by such a process could be replenished from aninternal or external reservoir.

Similar to the temperature actuation discussed above, chemical reactionsgenerating gaseous products may produce an increase in pressuresufficient for membrane actuation.

Networked Systems:

FIGS. 23A and 23B show a views of a single on/off valve, identical tothe systems set forth above, (for example in FIG. 7A). FIGS. 24A and 24Bshows a peristaltic pumping system comprised of a plurality of thesingle addressable on/off valves as seen in FIG. 23, but networkedtogether. FIG. 25 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIG. 24. FIGS.26A and 26B show a schematic view of a plurality of flow channels whichare controllable by a single control line. This system is also comprisedof a plurality of the single addressable on/off valves of FIG. 23,multiplexed together, but in a different arrangement than that of FIG.23. FIG. 27 is a schematic illustration of a multiplexing system adaptedto permit fluid flow through selected channels, comprised of a pluralityof the single on/off valves of FIG. 23, joined or networked together.

Referring first to FIGS. 23A and 23B, a schematic of flow channels 30and 32 is shown. Flow channel 30 preferably has a fluid (or gas) flow Fpassing therethrough. Flow channel 32, (which crosses over flow channel30, as was already explained herein), is pressurized such that membrane25 separating the flow channels may be depressed into the path of flowchannel 30, shutting off the passage of flow F therethrough, as has beenexplained. As such, “flow channel” 32 can also be referred to as a“control line” which actuates a single valve in flow channel 30. InFIGS. 23 to 26, a plurality of such addressable valves are joined ornetworked together in various arrangements to produce pumps, capable ofperistaltic pumping, and other fluidic logic applications.

Referring to FIGS. 24A and 24B, a system for peristaltic pumping isprovided, as follows. A flow channel 30 has a plurality of generallyparallel flow channels (i.e.: control lines) 32A, 32B and 32C passingthereover. By pressurizing control line 32A, flow F through flow channel30 is shut off under membrane 25A at the intersection of control line32A and flow channel 30.

Similarly, (but not shown), by pressurizing control line 32B, flow Fthrough flow channel 30 is shut off under membrane 25B at theintersection of control line 32B and flow channel 30, etc.

Each of control lines 32A, 32B, and 32C is separately addressable.Therefore, peristalsis may be actuated by the pattern of actuating 32Aand 32C together, followed by 32A, followed by 32A and 32B together,followed by 32B, followed by 32B and C together, etc. This correspondsto a successive “101, 100, 110, 010, 011, 001” pattern, where “0”indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns.

In experiments performed by the inventors, a pumping rate of 2.35 nL/swas measured by measuring the distance traveled by a column of water inthin (0.5 mm i.d.) tubing; with 100×100×10 μm valves under an actuationpressure of 40 kPa. The pumping rate increased with actuation frequencyuntil approximately 75 Hz, and then was nearly constant until above 200Hz. The valves and pumps are also quite durable and the elastomermembrane, control channels, or bond have never been observed to fail. Inexperiments performed by the inventors, none of the valves in theperistaltic pump described herein show any sign of wear or fatigue aftermore than 4 million actuations. In addition to their durability, theyare also gentle. A solution of E. Coli pumped through a channel andtested for viability showed a 94% survival rate.

FIG. 25 is a graph showing experimentally achieved pumping rates vs.frequency for the peristaltic pumping system of FIG. 24.

FIGS. 26A and 26B illustrates another way of assembling a plurality ofthe addressable valves of FIG. 21. Specifically, a plurality of parallelflow channels 30A, 30B, and 30C are provided. Flow channel (i.e.:control line) 32 passes thereover across flow channels 30A, 30B, and30C. Pressurization of control line 32 simultaneously shuts off flowsF1, F2 and F3 by depressing membranes 25A, 25B, and 25C located at theintersections of control line 32 and flow channels 30A, 30B, and 30C.

FIG. 27 is a schematic illustration of a multiplexing system adapted toselectively permit fluid to flow through selected channels, as follows.The downward deflection of membranes separating the respective flowchannels from a control line passing thereabove (for example, membranes25A, 25B, and 25C in FIGS. 26A and 26B) depends strongly upon themembrane dimensions. Accordingly, by varying the widths of flow channelcontrol line 32 in FIGS. 26A and 26B, it is possible to have a controlline pass over multiple flow channels, yet only actuate (i.e.: seal)desired flow channels. FIG. 27 illustrates a schematic of such a system,as follows.

A plurality of parallel flow channels 30A, 30B, 30C, 30D, 30E and 30Fare positioned under a plurality of parallel control lines 32A, 32B,32C, 32D, 32E and 32F. Control channels 32A, 32B, 32C, 32D, 32E and 32Fare adapted to shut off fluid flows F1, F2, F3, F4, F5 and F6 passingthrough parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F using anyof the valving systems described above, with the following modification.

Each of control lines 32A, 32B, 32C, 32D, 32E and 32F have both wide andnarrow portions. For example, control line 32A is wide in locationsdisposed over flow channels 30A, 30C and 30E. Similarly, control line32B is wide in locations disposed over flow channels 30B, 30D and 30F,and control line 32C is wide in locations disposed over flow channels30A, 30B, 30E and 30F.

At the locations where the respective control line is wide, itspressurization will cause the membrane (25) separating the flow channeland the control line to depress significantly into the flow channel,thereby blocking the flow passage therethrough. Conversely, in thelocations where the respective control line is narrow, membrane (25)will also be narrow. Accordingly, the same degree of pressurization willnot result in membrane (25) becoming depressed into the flow channel(30). Therefore, fluid passage thereunder will not be blocked.

For example, when control line 32A is pressurized, it will block flowsF1, F3 and F5 in flow channels 30A, 30C and 30E. Similarly, when controlline 32C is pressurized, it will block flows F1, F2, F5 and F6 in flowchannels 30A, 30B, 30E and 30F. As can be appreciated, more than onecontrol line can be actuated at the same time. For example, controllines 32A and 32C can be pressurized simultaneously to block all fluidflow except F4 (with 32A blocking F1, F3 and F5; and 32C blocking F1,F2, F5 and F6).

By selectively pressurizing different control lines (32) both togetherand in various sequences, a great degree of fluid flow control can beachieved. Moreover, by extending the present system to more than sixparallel flow channels (30) and more than four parallel control lines(32), and by varying the positioning of the wide and narrow regions ofthe control lines, very complex fluid flow control systems may befabricated. A property of such systems is that it is possible to turn onany one flow channel out of n flow channels with only 2(log₂n) controllines.

The inventors have succeeded in fabricating microfluidic structures withdensities of 30 devices/mm², but greater densities are achievable. Forexample, FIG. 60 shows a plan view of multiplexer device 6500 comprisingsixty-four parallel flow channels 6502 controlled by twelve overlyingcontrol channels 6504. Fluid is introduced into multiplexer structure6500 through input 6506, and the distributed to flow channels 6502through the peristaltic pumping action of pumping control lines 6508a-c.

Control channels 6504 a-1 may be understood as six pairs of controllines 6504 a-b, 6504 c-d, 6504 e-f, 6504 g-h, 6504 i-j, and 6504 k-1featuring complementary wide and narrow channel regions. For example,application of a control pressure to line 6504 a will close the firstthirty-two flow channels 6502, while application of a control pressureto line 6504 b will close the other thirty-two flow channels 6502.Similarly, application of a control pressure to line 6504 k will closeone set of alternating flow channels 6502, while application of acontrol pressure to line 65041 will close the other set of alternatingflow channels 6502. Thus by selectively applying a control pressure toone line of each control line pairs 6504 a-b, 6504 c-d, 6504 e-f, 6504g-h, 6504 i-j, and 6504 k-1, the flow of fluid from output 6510 ofmultiplexer structure 6500 can be limited to a single control channel.

While the above description illustrates a multiplexer having sixty-fourflow channels, this is merely one particular embodiment. A multiplexerstructure in accordance with the present invention is not limited to anyparticular number of flow channels. A non-exclusive list of the numberof flow channels for a multiplexer structure in accordance with thepresent invention follows: four, eight, sixteen, thirty-two, sixty-four,ninety-six, one hundred and twenty-eight, two hundred and fifty-six,three hundred and eighty-four, five hundred and twelve, one thousandtwenty-four, and one thousand five hundred and thirty-six.

Multiplexer structures comprising number of flow channels other thanthose listed are also contemplated, so long as the 2(log₂n) relationgoverning the minimum number of control lines for a given number of flowchannels is satisfied. Multiplexer structures having less than 2(log₂n)control lines for n flow lines are also contemplated by the presentinvention. Such multiplexer structures could generate useful actuationpatterns (i.e. 1111000011110000 . . . ) in the flow lines.

Selectively Addressable Reaction Chambers Along Flow Lines:

In a further embodiment of the invention, illustrated in FIGS. 28A, 28B,28C and 28D, a system for selectively directing fluid flow into one moreof a plurality of reaction chambers disposed along a flow line isprovided.

FIG. 28A shows a top view of a flow channel 30 having a plurality ofreaction chambers 80A and 80B disposed therealong. Preferably flowchannel 30 and reaction chambers 80A and 80B are formed together asrecesses into the bottom surface of a first layer 100 of elastomer.

FIG. 28B shows a bottom plan view of another elastomeric layer 110 withtwo control lines 32A and 32B each being generally narrow, but havingwide extending portions 33A and 33B formed as recesses therein.

As seen in the exploded view of FIG. 28C, and assembled view of FIG.28D, elastomeric layer 110 is placed over elastomeric layer 100. Layers100 and 110 are then bonded together, and the integrated system operatesto selectively direct fluid flow F (through flow channel 30) into eitheror both of reaction chambers 80A and 80B, as follows. Pressurization ofcontrol line 32A will cause the membrane 25 (i.e.: the thin portion ofelastomer layer 100 located below extending portion 33A and over regions82A of reaction chamber 80A) to become depressed, thereby shutting offfluid flow passage in regions 82A, effectively sealing reaction chamber80 from flow channel 30. As can also be seen, extending portion 33A iswider than the remainder of control line 32A. As such, pressurization ofcontrol line 32A will not result in control line 32A sealing flowchannel 30.

As can be appreciated, either or both of control lines 32A and 32B canbe actuated at once. When both control lines 32A and 32B are pressurizedtogether, sample flow in flow channel 30 will enter neither of reactionchambers 80A or 80B.

The concept of selectably controlling fluid introduction into variousaddressable reaction chambers disposed along a flow line (FIG. 28) canbe combined with concept of selectably controlling fluid flow throughone or more of a plurality of parallel flow lines (FIG. 27) to yield asystem in which a fluid sample or samples can be can be sent to anyparticular reaction chamber in an array of reaction chambers. An exampleof such a system is provided in FIG. 29, in which parallel controlchannels 32A, 32B and 32C with extending portions 34 (all shown inphantom) selectively direct fluid flows F1 and F2 into any of the arrayof reaction wells 80A, 80B, 80C or 80D as explained above; whilepressurization of control lines 32C and 32D selectively shuts off flowsF2 and F1, respectively.

In yet another novel embodiment, fluid passage between parallel flowchannels is possible. Referring to FIG. 30, either or both of controllines 32A or 32D can be depressurized such that fluid flow throughlateral passageways 35 (between parallel flow channels 30A and 30B) ispermitted. In this aspect of the invention, pressurization of controllines 32C and 32D would shut flow channel 30A between 35A and 35B, andwould also shut lateral passageways 35B. As such, flow entering as flowF1 would sequentially travel through 30A, 35A and leave 30B as flow F4.

Switchable Flow Arrays

In yet another novel embodiment, fluid passage can be selectivelydirected to flow in either of two perpendicular directions. An exampleof such a “switchable flow array” system is provided in FIGS. 31A to31D. FIG. 31A shows a bottom view of a first layer of elastomer 90, (orany other suitable substrate), having a bottom surface with a pattern ofrecesses forming a flow channel grid defined by an array of solid posts92, each having flow channels passing therearound.

In preferred aspects, an additional layer of elastomer is bound to thetop surface of layer 90 such that fluid flow can be selectively directedto move either in direction F1, or perpendicular direction F2. FIG. 31is a bottom view of the bottom surface of the second layer of elastomer95 showing recesses formed in the shape of alternating “vertical”control lines 96 and “horizontal” control lines 94. “Vertical” controllines 96 have the same width therealong, whereas “horizontal” controllines 94 have alternating wide and narrow portions, as shown.

Elastomeric layer 95 is positioned over top of elastomeric layer 90 suchthat “vertical” control lines 96 are positioned over posts 92 as shownin FIG. 31C and “horizontal” control lines 94 are positioned with theirwide portions between posts 92, as shown in FIG. 31D.

As can be seen in FIG. 31C, when “vertical” control lines 96 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 98 will be deflected downwardly over the array of flow channelssuch that flow in only able to pass in flow direction F2 (i.e.:vertically), as shown.

As can be seen in FIG. 31D, when “horizontal” control lines 94 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 99 will be deflected downwardly over the array of flow channels,(but only in the regions where they are widest), such that flow in onlyable to pass in flow direction F1 (i.e.: horizontally), as shown.

The design illustrated in FIG. 31 allows a switchable flow array to beconstructed from only two elastomeric layers, with no vertical viaspassing between control lines in different elastomeric layers required.If all vertical flow control lines 94 are connected, they may bepressurized from one input. The same is true for all horizontal flowcontrol lines 96.

Biopolymer Synthesis

The present elastomeric valving structures can also be used inbiopolymer synthesis, for example, in synthesizing oligonucleotides,proteins, peptides, DNA, etc. In a preferred aspect, such biopolymersynthesis systems may comprise an integrated system comprising an arrayof reservoirs, fluidic logic (according to the present invention) forselecting flow from a particular reservoir, an array of channels orreservoirs in which synthesis is performed, and fluidic logic (alsoaccording to the present invention) for determining into which channelsthe selected reagent flows. An example of such a system 200 isillustrated in FIG. 32, as follows.

Four reservoirs 150A, 150B, 150C and 150D have bases A, C, T and Grespectively disposed therein, as shown. Four flow channels 30A, 30B,30C and 30D are connected to reservoirs 150A, 150B, 150C and 150D. Fourcontrol lines 32A, 32B, 32C and 32D (shown in phantom) are disposedthereacross with control line 32A permitting flow only through flowchannel 30A (i.e.: sealing flow channels 30B, 30C and 30D), when controlline 32A is pressurized. Similarly, control line 32B permits flow onlythrough flow channel 30B when pressurized. As such, the selectivepressurization of control lines 32A, 32B, 32C and 32D sequentiallyselects a desired base A, C, T and G from a desired reservoir 150A,150B, 150C or 150D. The fluid then passes through flow channel 120 intoa multiplexed channel flow controller 125, (including, for example, anysystem as shown in FIGS. 26A to 31D) which in turn directs fluid flowinto one or more of a plurality of synthesis channels or chambers 122A,122B, 122C, 122D or 122E in which solid phase synthesis may be carriedout.

FIG. 33 shows a further extension of this system on which a plurality ofreservoirs R1 to R13 (which may contain bases A, T, C and G, or anyother reactants, such as would be used in combinatorial chemistry), areconnected to systems 200 as set forth in FIG. 32. Systems 200 areconnected to a multiplexed channel flow controller 125, (including, forexample, any system as shown in FIGS. 26A to 31D) which is in turnconnected to a switchable flow array (for example as shown in FIG. 31).An advantage of this system is that both of multiplexed channel flowcontrollers 125 and fluid selection systems 200 can be controlled by thesame pressure inputs 170 and 172, provided a “close horizontal” and a“close vertical” control lines (160 and 162, in phantom) are alsoprovided.

In further alternate aspects of the invention, a plurality ofmultiplexed channel flow controllers (such as 125) may be used, witheach flow controller initially positioned stacked above one another on adifferent elastomeric layer, with vertical vias or interconnects betweenthe elastomer layers (which may be created by lithographicallypatterning an etch resistant layer on top of a elastomer layer, thenetching the elastomer and finally removing the etch resist before addingthe last layer of elastomer).

For example, a vertical via in an elastomer layer can be created byetching a hole down onto a raised line on a micromachined mold, andbonding the next layer such that a channel passes over that hole. Inthis aspect of the invention, multiple synthesis with a plurality ofmultiplexed channel flow controllers 125 is possible.

The bonding of successive layers of molded elastomer to form amulti-layer structure is shown in FIG. 34, which is an opticalmicrograph of a section of a test structure composed of seven layers ofelastomer. The scale bar of FIG. 34 is 200 μm.

One method for fabricating an elastomer layer having the vertical viafeature utilized in a multi-layer structure is shown in FIGS. 35A-35D.FIG. 35A shows formation of elastomer layer 3500 over micromachined mold3502 including raised line 3502 a.

FIG. 35B shows formation of metal etch blocking layer 3504 overelastomer layer 3500, followed by the patterning of photoresist mask3506 over etch blocking layer 3504 to cover masked regions 3508 andleave exposed unmasked regions 3510. FIG. 35C shows the exposure tosolvent which removes etch blocking layer 3504 in unmasked regions 3510.

FIG. 35D shows removal of the patterned photoresist, followed bysubsequent etching of underlying elastomer 3500 in unmasked regions 3510to form vertical via 3512. Subsequent exposure to solvent removesremaining etch blocking layer 3504 in masked regions 3508 selective tothe surrounding elastomer 3500 and mold 3502. This elastomer layer maythen be incorporated into an elastomer structure by multilayer softlithography.

This series of steps can be repeated as necessary to form amulti-layered structure having the desired number and orientation ofvertical vias between channels of successive elastomer layers.

The inventors of the present invention have succeeded in etching viasthrough GE RTV 615 layers using a solution of Tetrabutylammoniumfluoride in organic solvent. Gold serves as the etch blocking material,with gold removed selective to GE RTV 615 utilizing a KI/I₂/H₂O mixture.

Alternatively, vertical vias between channels in successive elastomerlayers could be formed utilizing a negative mask technique. In thisapproach, a negative mask of a metal foil is patterned, and subsequentformation of an etch blocking layer is inhibited where the metal foil ispresent. Once the etch blocking material is patterned, the negativemetal foil mask is removed, permitting selective etching of theelastomer as described above.

In yet another approach, vertical vias could be formed in an elastomerlayer using ablation of elastomer material through application ofradiation from an applied laser beam.

While the above approach is described in connection with the synthesisof biopolymers, the invention is not limited to this application. Thepresent invention could also function in a wide variety of combinatorialchemical synthesis approaches.

Other Applications:

Advantageous applications of the present monolithic microfabricatedelastomeric valves and pumps are numerous. Accordingly, the presentinvention is not limited to any particular application or use thereof.In preferred aspects, the following uses and applications for thepresent invention are contemplated.

1. Cell/DNA Sorting

The present microfluidic pumps and valves can also be used in flowcytometers for cell sorting and DNA sizing. Sorting of objects basedupon size is extremely useful in many technical fields.

For example, many assays in biology require determination of the size ofmolecular-sized entities. Of particular importance is the measurement oflength distribution of DNA molecules in a heterogeneous solution. Thisis commonly done using gel electrophoresis, in which the molecules areseparated by their differing mobility in a gel matrix in an appliedelectric field, and their positions detected by absorption or emissionof radiation. The lengths of the DNA molecules are then inferred fromtheir mobility.

While powerful, electrophoretic methods pose disadvantages. For mediumto large DNA molecules, resolution, i.e. the minimum length differenceat which different molecular lengths may be distinguished, is limited toapproximately 10% of the total length. For extremely large DNAmolecules, the conventional sorting procedure is not workable. Moreover,gel electrophoresis is a relatively lengthy procedure, and may requireon the order of hours or days to perform.

The sorting of cellular-sized entities is also an important task.Conventional flow cell sorters are designed to have a flow chamber witha nozzle and are based on the principle of hydrodynamic focusing withsheath flow. Most conventional cell sorters combine the technology ofpiezo-electric drop generation and electrostatic deflection to achievedroplet generation and high sorting rates. However, this approach offerssome important disadvantages. One disadvantage is that the complexity,size, and expense of the sorting device requires that it be reusable inorder to be cost-effective. Reuse can in turn lead to problems withresidual materials causing contamination of samples and turbulent fluidflow.

Therefore, there is a need in the art for a simple, inexpensive, andeasily fabricated sorting device which relies upon the mechanicalcontrol of fluid flow rather than upon electrical interactions betweenthe particle and the solute.

FIG. 36 shows one embodiment of a sorting device in accordance with thepresent invention. Sorting device 3600 is formed from a switching valvestructure created from channels present in an elastomeric block.Specifically, flow channel 3602 is T-shaped, with stem 3602 a of flowchannel 3602 in fluid communication with sample reservoir 3604containing sortable entities 3606 of different types denoted by shape(square, circle, triangle, etc.). Left branch 3602 b of flow channel3602 is in fluid communication with waste reservoir 3608. Right branch3602 c of flow channel 3602 is in communication with collectionreservoir 3610.

Control channels 3612 a, 3612 b, and 3612 c overlie and are separatedfrom stem 3602 a of flow channel 3602 by elastomeric membrane portions3614 a, 3614 b, and 3614 c respectively. Together, stem 3602 a of flowchannel 3602 and control channels 3612 a, 3612 b, and 3612 c form firstperistaltic pump structure 3616 similar to that described at lengthabove in connection with FIG. 24 a.

Control channel 3612 d overlies and is separated from right branch 3602c of flow channel 3602 by elastomeric membrane portion 3614 d. Together,right branch 3602 c of flow channel 3602 and control channels 3612 dforms first valve structure 3618 a. Control channel 3612 e overlies andis separated from left branch 3602 c of flow channel 3602 by elastomericmembrane portion 3614 e. Together, left branch 3602 c of flow channel3602 and control channel 3612 e forms second valve structure 3618 b.

As shown in FIG. 36, stem 3602 a of flow channel 3602 narrowsconsiderably as it approaches detection widow 3620 adjacent to thejunction of stem 3602 a, right branch 3602 b, and left branch 3602 c.Detection window 3620 is of sufficient width to allow for uniformillumination of this region. In one embodiment, the width of the stemnarrows from 100 μm to 5 μm at the detection window. The width of thestem at the detection window can be precisely formed using the softlithography or photoresist encapsulation fabrication techniquesdescribed extensively above, and will be depend upon the nature and sizeof the entity to be sorted.

Operation of sorting device in accordance with one embodiment of thepresent invention is as follows.

The sample is diluted to a level such that only a single sortable entitywould be expected to be present in the detection window at any time.Peristaltic pump 3616 is activated by flowing a fluid through controlchannels 3612 a-c as described extensively above. In addition, secondvalve structure 3618 b is closed by flowing fluid through controlchannel 3612 e. As a result of the pumping action of peristaltic pump3616 and the blocking action of second valve 3618 b, fluid flows fromsample reservoir 3604 through detection window 3620 into waste reservoir3608. Because of the narrowing of stem 3604, sortable entities presentin sample reservoir 3604 are carried by this regular fluid flow, one ata time, through detection window 3620.

Radiation 3640 from source 3642 is introduced into detection window3620. This is possible due to the transmissive property of theelastomeric material. Absorption or emission of radiation 3640 bysortable entity 3606 is then detected by detector 3644.

If sortable entity 3606 a within detection window 3620 is intended to besegregated and collected by sorting device 3600, first valve 3618 a isactivated and second valve 3618 b is deactivated. This has the effect ofdrawing sortable entity 3606 a into collection reservoir 3610, and atthe same time transferring second sortable entity 3606 b into detectionwindow 3620. If second sortable entity 3602 b is also identified forcollection, peristaltic pump 3616 continues to flow fluid through rightbranch 3602 c of flow channel 3602 into collection reservoir 3610.However, if second entity 3606 b is not to be collected, first valve3618 a opens and second valve 3618 b closes, and first peristaltic pump3616 resumes pumping liquid through left branch 3602 b of flow channel3602 into waste reservoir 3608.

While one specific embodiment of a sorting device and a method foroperation thereof is described in connection with FIG. 36, the presentinvention is not limited to this embodiment. For example, fluid need notbe flowed through the flow channels using the peristaltic pumpstructure, but could instead be flowed under pressure with theelastomeric valves merely controlling the directionality of flow. In yetanother embodiment, a plurality of sorting structures could be assembledin series in order to perform successive sorting operations, with thewaste reservoir of FIG. 36 simply replaced by the stem of the nextsorting structure.

Moreover, a high throughput method of sorting could be employed, whereina continuous flow of fluid from the sample reservoir through the windowand junction into the waste reservoir is maintained until an entityintended for collection is detected in the window. Upon detection of anentity to be collected, the direction of fluid flow by the pumpstructure is temporarily reversed in order to transport the desiredparticle back through the junction into the collection reservoir. Inthis manner, the sorting device could utilize a higher flow rate, withthe ability to backtrack when a desired entity is detected. Such analternative high throughput sorting technique could be used when theentity to be collected is rare, and the need to backtrack infrequent.

Sorting in accordance with the present invention would avoid thedisadvantages of sorting utilizing conventional electrokinetic flow,such as bubble formation, a strong dependence of flow magnitude anddirection on the composition of the solution and surface chemistryeffects, a differential mobility of different chemical species, anddecreased viability of living organisms in the mobile medium.

2. Semiconductor Processing

Systems for semiconductor gas flow control, (particularly for epitaxialapplications in which small quantities of gases are accurately metered),are also contemplated by the present invention. For example, duringfabrication of semiconductor devices solid material is deposited on topof a semiconductor substrate utilizing chemical vapor deposition (CVD).This is accomplished by exposing the substrate to a mixture of gasprecursor materials, such that these gases react and the resultingproduct crystallizes on top of the substrate.

During such CVD processes, conditions must be carefully controlled toensure uniform deposition of material free of defects that could degradethe operation of the electrical device. One possible source ofnonuniformity is variation in the flow rate of reactant gases to theregion over the substrate. Poor control of the gas flow rate can alsolead to variations in the layer thicknesses from run to run, which isanother source of error. Unfortunately, there has been a significantproblem in controlling the amount of gas flowed into the processingchamber, and maintaining stable flow rates in conventional gas deliverysystems.

Accordingly, FIG. 37A shows one embodiment of the present inventionadapted to convey, at precisely-controllable flow rates, processing gasover the surface of a semiconductor wafer during a CVD process.Specifically, semiconductor wafer 3700 is positioned upon wafer support3702 located within a CVD chamber. Elastomeric structure 3704 containinga large number of evenly distributed orifices 3706 is positioned justabove the surface of wafer 3700.

A variety of process gases are flowed at carefully controlled rates fromreservoirs 3708 a and 3708 b, through flow channels in elastomeric block3704, and out of orifices 3706. As a result of the precisely controlledflow of process gases above wafer 3700, solid material 3710 having anextremely uniform structure is deposited.

Precise metering of reactant gas flow rates utilizing valve and/or pumpstructures of the present invention is possible for several reasons.First, gases can be flowed through valves that respond in a linearfashion to an applied actuation pressure, as is discussed above inconnection with FIGS. 21A and 21B. Alternatively or in addition tometering of gas flow using valves, the predictable behavior of pumpstructures in accordance with the present invention can be used toprecisely meter process gas flow.

In addition to the chemical vapor deposition processes described above,the present technique is also useful to control gas flow in techniquessuch as molecular beam epitaxy and reactive ion etching.

3. Micro Mirror Arrays

While the embodiments of the present invention described thus far relateto operation of a structure composed entirely of elastomeric material,the present invention is not limited to this type of structure.Specifically, it is within the scope of the present invention to combinean elastomeric structure with a conventional, silicon-basedsemiconductor structure.

For example, further contemplated uses of the present microfabricatedpumps and valves are in optical displays in which the membrane in anelastomeric structure reflects light either as a flat planar or as acurved surface depending upon whether the membrane is activated. Assuch, the membrane acts as a switchable pixel. An array of suchswitchable pixels, with appropriate control circuitry, could be employedas a digital or analog micro mirror array.

Accordingly, FIG. 38 shows an exploded view of a portion of oneembodiment of a micro mirror array in accordance with the presentinvention.

Micro mirror array 3800 includes first elastomer layer 3802 overlyingand separated from and underlying semiconductor structure 3804 by secondelastomer layer 3806. Surface 3804 a of semiconductor structure 3804bears a plurality of electrodes 3810. Electrodes 3810 are individuallyaddressable through conducting row and column lines, as would be knownto one of ordinary skill in the art.

First elastomeric layer 3802 includes a plurality of intersectingchannels 3822 underlying an electrically conducting, reflectingelastomeric membrane portion 3802 a. First elastomeric layer 3802 isaligned over second elastomeric layer 3806 and underlying semiconductordevice 3804 such that points of intersection of channels 3822 overlieelectrodes 3810.

In one embodiment of a method of fabrication in accordance with thepresent invention, first elastomeric layer 3822 may be formed byspincoating elastomeric material onto a mold featuring intersectingchannels, curing the elastomer, removing the shaped elastomer from themold, and introducing electrically conducting dopant into surface regionof the shaped elastomer. Alternatively as described in connection withFIGS. 7C-7G above, first elastomeric layer 3822 may be formed from twolayers of elastomer by inserting elastomeric material into a moldcontaining intersecting channels such that the elastomeric material isflush with the height of the channel walls, and then bonding a separatedoped elastomer layer to the existing elastomeric material to form amembrane on the top surface.

Alternatively, the first elastomeric layer 3802 may be produced fromelectrically conductive elastomer, where the electrical conductivity isdue either to doping or to the intrinsic properties of the elastomermaterial.

During operation of reflecting structure 3800, electrical signals arecommunicated along a selected row line and column line to electrode 3810a. Application of voltage to electrode 3810 a generates an attractiveforce between electrode 3810 a and overlying membrane 3802 a.

This attractive force actuates a portion of membrane 3802 a, causingthis membrane portion to flex downward into the cavity resulting fromintersection of the channels 3822. As a result of distortion of membrane3802 a from planar to concave, light is reflected differently at thispoint in the surface of elastomer structure 3802 than from thesurrounding planar membrane surface. A pixel image is thereby created.

The appearance of this pixel image is variable, and may be controlled byaltering the magnitude of voltage applied to the electrode. A highervoltage applied to the electrode will increase the attractive force onthe membrane portion, causing further distortion in its shape. A lowervoltage applied to the electrode will decrease the attractive force onthe membrane, reducing distortion in its shape from the planar. Eitherof these changes will affect the appearance of the resulting pixelimage.

A variable micro mirror array structure as described could be used in avariety of applications, including the display of images. Anotherapplication for a variable micro mirror array structure in accordancewith an embodiment of the present invention would be as a high capacityswitch for a fiber optics communications system, with each pixel capableof affecting the reflection and transfer of a component of an incidentlight signal.

While the above embodiment describes a composite,elastomer/semiconductor structure that is utilized as a miromirrorarray, the present invention is not limited to this particularembodiment.

For example, another optical application for embodiments of the presentinvention relate to switchable Bragg mirrors. Bragg mirrors reflect aspecific range of wavelengths of incident light, allowing all otherwavelengths to pass. A Bragg mirror in accordance with an embodiment ofthe present invention was fabricated by sputter depositing a mirrorcomprising thirty alternating 1 μm thick layers of SiO and Si₃N₄ over a30 μm thick RTV elastomer membrane, that in turn overlied a controlchannel having a depth of 10 μm and a width of 100 μm. Followingpassivation of the mirror surface with additional RTV elastomer,application of a control pressure of 15 psi to the control channelcaused the membrane to deform and allows certain wavelengths to pass.This result is shown in FIG. 65, which plots intensity of light at 490nm passing through the mirror versus switching cycle. A Bragg mirror inaccordance with embodiments of the present invention could be utilizedfor a variety of purposes, including optical filtering.

4. Refracting Structures

The micro-mirror array structure just described controls reflection ofincident light. However, the present invention is not limited tocontrolling reflection. Yet another embodiment of the present inventionenables the exercise of precise control over refraction of incidentlight in order to create lens and filter structures.

FIG. 39 shows one embodiment of a refractive structure in accordancewith the present invention. Refractive structure 3900 includes firstelastomeric layer 3902 and second elastomeric layer 3904 composed ofelastomeric material capable of transmitting incident light 3906.

First elastomeric layer 3902 has convex portion 3902 a which may becreated by curing elastomeric material formed over a micromachined moldhaving a concave portion. Second elastomeric layer 3904 has a flowchannel 3905 and may be created from a micromachined mold having araised line as discussed extensively above.

First elastomer layer 3902 is bonded to second elastomer layer 3904 suchthat convex portion 3902 a is positioned above flow channel 3905. Thisstructure can serve a variety of purposes.

For example, light incident to elastomeric structure 3900 would befocused into the underlying flow channel, allowing the possibleconduction of light through the flow channel. Alternatively, in oneembodiment of an elastomeric device in accordance with the presentinvention, fluorescent or phosphorescent liquid could be flowed throughthe flow channel, with the resulting light from the fluid refracted bythe curved surface to form a display.

FIG. 40 shows another embodiment of a refractive structure in accordancewith the present invention. Refractive structure 4000 is a multilayeroptical train based upon a Fresnel lens design. Specifically, refractivestructure 4000 is composed of four successive elastomer layers 4002,4004, 4006, and 4008, bonded together. The upper surfaces of each offirst, second, and third elastomer layers 4002, 4004, and 4006 bearuniform serrations 4010 regularly spaced by a distance X that is muchlarger than the wavelength of the incident light. Serrations 4010 serveto focus the incident light, and may be formed through use of amicromachined mold as described extensively above. First, second, andthird elastomer layers 4002, 4004, and 4006 function as Fresnel lensesas would be understood of one of ordinary skill in the art.

Fourth elastomeric layer 4008 bears uniform serrations 4012 having amuch smaller size than the serrations of the overlying elastomericlayers. Serrations 4012 are also spaced apart by a much smaller distanceY than the serrations of the overlying elastomeric layers, with Y on theorder of the wavelength of incident light. such that elastomeric layer4008 functions as a diffraction grating.

FIG. 41 illustrates an embodiment of a refractive structure inaccordance with the present invention which utilizes difference inmaterial refractive index to primarily accomplish diffraction.Refractive structure 4100 includes lower elastomeric portion 4102covered by upper elastomeric portion 4104. Both lower elastomericportion 4102 and upper elastomeric portion 4104 are composed of materialtransmitting incident light 4106. Lower elastomeric portion 4102includes a plurality of serpentine flow channels 4108 separated byelastomeric lands 4110. Flow channels 4108 include fluid 4112 having adifferent refractive index than the elastomeric material making up lands4110. Fluid 4112 is pumped through serpentine flow channels 4108 by theoperation of pump structure 4114 made up of parallel control channels4116 a and 4116 b overlying and separated from inlet portion 4108 a offlow channel 4108 by moveable membrane 4118.

Light 4106 incident to refractive structure 4100 encounters a series ofuniformly-spaced fluid-filled flow channels 4108 separated byelastomeric lands 4110. As a result of the differing optical propertiesof material present in these respective fluid/elastomer regions,portions of the incident light are not uniformly refracted and interactto form an interference pattern. A stack of refractive structures of themanner just described can accomplish even more complex and specializedrefraction of incident light.

The refractive elastomeric structures just described can fulfill avariety of purposes. For example, the elastomeric structure could act asa filter or optical switch to block selected wavelengths of incidentlight. Moreover, the refractive properties of the structure could bereadily adjusted depending upon the needs of a particular application.

For example, the composition (and hence refractive index) of fluidflowed through the flow channels could be changed to affect diffraction.Alternatively, or in conjunction with changing the identity of the fluidflowed, the distance separating adjacent flow channels can be preciselycontrolled during fabrication of the structure in order to generate anoptical interference pattern having the desired characteristics.

Yet another application involving refraction by embodiments inaccordance with the present invention is a tunable microlens structure.As shown in FIG. 66, tunable microlens structure 7100 comprises chamber7102 connected to pneumatic control line 7104. Chamber 7102 may beformed by soft lithography of a transparent substrate such as RTV, whichis then bonded to elastomer membrane 7106. Chamber 7102 is then filledwith a fluid 7103 chosen for its index of refraction. When control line7104 line is pressurized, membrane 7106 expands in a bulb over chamber7102, creating a lens. Radius R of curvature of membrane 7106, and hencethe focal length of the lens, may be controlled by varying the pressurein control line 7104 and therefore tuning the lens. Possibleapplications of such a lens might be the separate interrogation ofdifferent channel layers, or the scanning of a bulk sample. Sincedefining the lens structure is accomplished using photolithography, suchlenses may be fabricated inexpensively and may be densely integrated ona fluidics device. Lenses may be made in a wide range of sizes, withdiameters varying from 1 μm to 1 cm. Alignment issues are significantlyreduced since lateral alignment is achieved by lithography, and verticalalignment is achieved by tuning.

5. Normally-Closed Valve Structure

FIGS. 7B and 7H above depict a valve structure in which the elastomericmembrane is moveable from a first relaxed position to a second actuatedposition in which the flow channel is blocked. However, the presentinvention is not limited to this particular valve configuration.

FIGS. 42A-42J show a variety of views of a normally-closed valvestructure in which the elastomeric membrane is moveable from a firstrelaxed position blocking a flow channel, to a second actuated positionin which the flow channel is open, utilizing a negative controlpressure.

FIG. 42A shows a plan view, and FIG. 42B shows a cross sectional viewalong line 42B-42B′, of normally-closed valve 4200 in an unactuatedstate. Flow channel 4202 and control channel 4204 are formed inelastomeric block 4206 overlying substrate 4205. Flow channel 4202includes a first portion 4202 a and a second portion 4202 b separated byseparating portion 4208. Control channel 4204 overlies separatingportion 4208. As shown in FIG. 42B, in its relaxed, unactuated position,separating portion 4008 remains positioned between flow channel portions4202 a and 4202 b, interrupting flow channel 4202.

FIG. 42C shows a cross-sectional view of valve 4200 wherein separatingportion 4208 is in an actuated position. When the pressure withincontrol channel 4204 is reduced to below the pressure in the flowchannel (for example by vacuum pump), separating portion 4208experiences an actuating force drawing it into control channel 4204. Asa result of this actuation force membrane 4208 projects into controlchannel 4204, thereby removing the obstacle to a flow of materialthrough flow channel 4202 and creating a passageway 4203. Upon elevationof pressure within control channel 4204, separating portion 4208 willassume its natural position, relaxing back into and obstructing flowchannel 4202.

The behavior of the membrane in response to an actuation force may bechanged by varying the width of the overlying control channel.Accordingly, FIGS. 42D-42H show plan and cross-sectional views of analternative embodiment of a normally-closed valve 4201 in which controlchannel 4207 is substantially wider than separating portion 4208. Asshown in cross-sectional views FIG. 42E-F along line 42E-42E′ of FIG.42D, because a larger area of elastomeric material is required to bemoved during actuation, the actuation force necessary to be applied isreduced.

FIGS. 42G and H show a cross-sectional views along line 40G-40G′ of FIG.40D. In comparison with the unactuated valve configuration shown in FIG.42G, FIG. 42H shows that reduced pressure within wider control channel4207 may under certain circumstances have the unwanted effect of pullingunderlying elastomer 4206 away from substrate 4205, thereby creatingundesirable void 4212.

Accordingly, FIG. 42I shows a plan view, and 42J a cross-sectional viewalong line 42J-42J′ of FIG. 42I, of valve structure 4220 which avoidsthis problem by featuring control line 4204 with a minimum width exceptin segment 4204 a overlapping separating portion 4208. As shown in FIG.42J, even under actuated conditions the narrower cross-section ofcontrol channel 4204 reduces the attractive force on the underlyingelastomer material 4206, thereby preventing this elastomer material frombeing drawn away from substrate 4205 and creating an undesirable void.

While a normally-closed valve structure actuated in response to pressureis shown in FIGS. 42A-42J, a normally-closed valve in accordance withthe present invention is not limited to this configuration. For example,the separating portion obstructing the flow channel could alternativelybe manipulated by electric or magnetic fields, as described extensivelyabove.

6. Separation of Materials

In a further application of the present invention, an elastomericstructure can be utilized to perform separation of materials. FIG. 43shows one embodiment of such a device.

Separation device 4300 features an elastomeric block 4301 includingfluid reservoir 4302 in communication with flow channel 4304. Fluid ispumped from fluid reservoir 4306 through flow channel 4308 byperistaltic pump structure 4310 formed by control channels 4312overlying flow channel 4304, as has been previously described at length.Alternatively, where a peristaltic pump structure in accordance with thepresent invention is unable to provide sufficient back pressure, fluidfrom a reservoir positioned outside the elastomeric structure may bepumped into the elastomeric device utilizing an external pump.

Flow channel 4304 leads to separation column 4314 in the form of achannel packed with separation matrix 4316 behind porous frit 4318. Asis well known in the art of chromatography, the composition of theseparation matrix 4316 depends upon the nature of the materials to beseparated and the particular chromatography technique employed. Theelastomeric separation structure is suitable for use with a variety ofchromatographic techniques, including but not limited to gel exclusion,gel permeation, ion exchange, reverse phase, hydrophobic interaction,affinity chromatography, fast protein liquid chromatography (FPLC) andall formats of high pressure liquid chromatography (HPLC). The highpressures utilized for HPLC may require the use of urethane,dicyclopentadiene or other elastomer combinations.

Samples are introduced into the flow of fluid into separation column4314 utilizing load channel 4319. Load channel 4319 receives fluidpumped from sample reservoir 4320 through pump 4321. Upon opening ofvalve 4322 and operation of pump 4321, sample is flowed from loadchannel 4319 into flow channel 4304. The sample is then flowed throughseparation column 4314 by the action of pump structure 4312. As a resultof differential mobility of the various sample components in separationmatrix 4316, these sample components become separated and are elutedfrom column 4314 at different times.

Upon elution from separation column 4314, the various sample componentspass into detection region 4324. As is well known in the art ofchromatography, the identity of materials eluted into detection region4324 can be determined utilizing a variety of techniques, including butnot limited to fluorescence, UV/visible/IR spectroscopy, radioactivelabeling, amperometric detection, mass spectroscopy, and nuclearmagnetic resonance (NMR).

A separation device in accordance with the present invention offers theadvantage of extremely small size, such that only small volumes of fluidand sample are consumed during the separation. In addition, the deviceoffers the advantage of increased sensitivity. In conventionalseparation devices, the size of the sample loop will prolong theinjection of the sample onto the column, causing width of the elutedpeaks to potentially overlap with one another. The extremely small sizeand capacity of the load channel in general prevents this peak diffusionbehavior from becoming a problem.

The separation structure shown in FIG. 43 represents only one embodimentof such a device, and other structures are contemplated by the presentinvention. For example, while the separation device of FIG. 43 featuresa flow channel, load loop, and separation column oriented in a singleplane, this is not required by the present invention. One or more of thefluid reservoir, the sample reservoir, the flow channel, the load loop,and the separation column could be oriented perpendicular to one anotherand/or to the plane of the elastomeric material utilizing via structureswhose formation is described at length above in connection with FIG.35A-D.

7. Cell Pen/Cell Cage/Cell Grinder

In yet a further application of the present invention, an elastomericstructure can be utilized to manipulate organisms or other biologicalmaterial. FIGS. 44A-44D show plan views of one embodiment of a cell penstructure in accordance with the present invention.

Cell pen array 4400 features an array of orthogonally-oriented flowchannels 4402, with an enlarged “pen” structure 4404 at the intersectionof alternating flow channels. Valve 4406 is positioned at the entranceand exit of each pen structure 4404. Peristaltic pump structures 4408are positioned on each horizontal flow channel and on the vertical flowchannels lacking a cell pen structure.

Cell pen array 4400 of FIG. 44A has been loaded with cells A-H that havebeen previously sorted, perhaps by a sorting structure as describedabove in conjunction with FIG. 36. FIGS. 44B-44C show the accessing andremoval of individually stored cell C by 1) opening valves 4406 oneither side of adjacent pens 4404 a and 4404 b, 2) pumping horizontalflow channel 4402 a to displace cells C and G, and then 3) pumpingvertical flow channel 4402 b to remove cell C. FIG. 44D shows thatsecond cell G is moved back into its prior position in cell pen array4400 by reversing the direction of liquid flow through horizontal flowchannel 4402 a.

The cell pen array 4404 described above is capable of storing materialswithin a selected, addressable position for ready access. However,living organisms such as cells may require a continuous intake of foodsand expulsion of wastes in order to remain viable. Accordingly, FIGS.45A and 45B show plan and cross-sectional views (along line 45B-45B′)respectively, of one embodiment of a cell cage structure in accordancewith the present invention.

Cell cage 4500 is formed as an enlarged portion 4500 a of a flow channel4501 in an elastomeric block 4503 in contact with substrate 4505. Cellcage 4500 is similar to an individual cell pen as described above inFIGS. 44A-44D, except that ends 4500 b and 4500 c of cell cage 4500 donot completely enclose interior region 4500 a. Rather, ends 4500 a and4500 b of cage 4500 are formed by a plurality of retractable pillars4502. Pillars 4502 may be part of a membrane structure of anormally-closed valve structure as described extensively above inconnection with FIGS. 42A-42J.

Specifically, control channel 4504 overlies pillars 4502. When thepressure in control channel 4504 is reduced, elastomeric pillars 4502are drawn upward into control channel 4504, thereby opening end 4500 bof cell cage 4500 and permitting a cell to enter. Upon elevation ofpressure in control channel 4504, pillars 4502 relax downward againstsubstrate 4505 and prevent a cell from exiting cage 4500.

Elastomeric pillars 4502 are of a sufficient size and number to preventmovement of a cell out of cage 4500, but also include gaps 4508 whichallow the flow of nutrients into cage interior 4500 a in order tosustain cell(s) stored therein. Pillars 4502 on opposite end 4500 c aresimilarly configured beneath second control channel 4506 to permitopening of the cage and removal of the cell as desired.

Under certain circumstances, it may be desirable to grind/disrupt cellsor other biological materials in order to access component pieces.

Accordingly, FIGS. 46A and 46B show plan and cross sectional views(along line 46B-46B′) respectively, of one embodiment of cell grinderstructure 4600 in accordance with the present invention. Cell grinder4600 includes a system of interdigitated posts 4602 within flow channel4604 which close together upon actuation of integral membrane 4606 byoverlying control channel 4608. By closing together, posts 4602 crushmaterial present between them.

Posts 4602 may be spaced at intervals appropriate to disrupt entities(cells) of a given size. For disruption of cellular material, spacing ofposts 4602 at an interval of about 2 μm is appropriate. In alternativeembodiments of a cell grinding structure in accordance with the presentinvention, posts 4602 may be located entirely on the above-lyingmembrane, or entirely on the floor of the control channel.

The cross-flow channel architecture illustrated shown in FIGS. 44A-44Dcan be used to perform functions other than the cell pen just described.For example, the cross-flow channel architecture can be utilized inmixing applications.

This is shown in FIGS. 69A-B, which illustrate a plan view of mixingsteps performed by a microfabricated structure in accordance anotherembodiment of the present invention. Specifically, portion 7400 of amicrofabricated mixing structure comprises first flow channel 7402orthogonal to and intersecting with second flow channel 7404. Controlchannels 7406 overlie flow channels 7402 and 7404 and form valve pairs7408 a-b and 7408 c-d that surround each intersection 7412.

As shown in FIG. 69A, valve pair 7408 a-b is initially opened whilevalve pair 7408 c-d is closed, and fluid sample 7410 is flowed tointersection 7412 through flow channel 7402. Valve pair 7408 c-d is thenactuated, trapping fluid sample 7410 at intersection 7412.

Next, as shown in FIG. 69B, valve pairs 7408 a-b and 7408 c-d areopened, such that fluid sample 7410 is injected from intersection 7412into flow channel 7404 bearing a cross-flow of fluid. The process shownin FIGS. 69A-B can be repeated to accurately dispense any number offluid samples down cross-flow channel 7404.

8. Pressure Oscillator

In yet a further application of the present invention, an elastomericstructure can be utilized to create a pressure oscillator structureanalogous to oscillator circuits frequently employed in the field ofelectronics. FIG. 47 shows a plan view of one embodiment of such apressure oscillator structure.

Pressure oscillator 4700 comprises an elastomeric block 4702 featuringflow channel 4704 formed therein. Flow channel 4704 includes an initialportion 4704 a proximate to pressure source 4706, and a serpentineportion 4704 b distal from pressure source 4706. Initial portion 4704 ais in contact with via 4708 in fluid communication with control channel4710 formed in elastomeric block 4702 above the level of flow channel4704. At a location more distal from pressure source 4706 than via 4708,control channel 4710 overlies and is separated from flow channel 4704 byan elastomeric membrane, thereby forming valve 4712 as previouslydescribed.

Pressure oscillator structure 4700 operates as follows. Initially,pressure source 4706 provides pressure along flow channel 4704 andcontrol channel 4710 through via 4708. Because of the serpentine shapeof flow channel 4704 b, pressure is lower in region 4704 b as comparedwith flow channel 4710. At valve 4712, the pressure difference betweenserpentine flow channel portion 4704 b and overlying control channel4710 eventually causes the membrane of valve 4712 to project downwardinto serpentine flow channel portion 4704 b, closing valve 4712. Owingto the continued operation of pressure source 4706 however, pressurebegins to build up in serpentine flow channel portion 4704 b behindclosed valve 4712. Eventually the pressure equalizes between controlchannel 4710 and serpentine flow channel portion 4704 b, and valve 4712opens.

Given the continuos operation of the pressure source, theabove-described build up and release of pressure will continueindefinitely, resulting in a regular oscillation of pressure. Such apressure oscillation device may perform any number of possiblefunctions, including but not limited to timing.

9. Side-Actuated Valve

While the above description has focused upon microfabricated elastomericvalve structures in which a control channel is positioned above andseparated by an intervening elastomeric membrane from an underlying flowchannel, the present invention is not limited to this configuration.FIGS. 48A and 48B show plan views of one embodiment of a side-actuatedvalve structure in accordance with one embodiment of the presentinvention.

FIG. 48A shows side-actuated valve structure 4800 in an unactuatedposition. Flow channel 4802 is formed in elastomeric layer 4804. Controlchannel 4806 abutting flow channel 4802 is also formed in elastomericlayer 4804. Control channel 4806 is separated from flow channel 4802 byelastomeric membrane portion 4808. A second elastomeric layer (notshown) is bonded over bottom elastomeric layer 4804 to enclose flowchannel 4802 and control channel 4806.

FIG. 48B shows side-actuated valve structure 4800 in an actuatedposition. In response to a build up of pressure within control channel4806, membrane 4808 deforms into flow channel 4802, blocking flowchannel 4802. Upon release of pressure within control channel 4806,membrane 4808 would relax back into control channel 4806 and open flowchannel 4802.

While a side-actuated valve structure actuated in response to pressureis shown in FIGS. 48A and 48B, a side-actuated valve in accordance withthe present invention is not limited to this configuration. For example,the elastomeric membrane portion located between the abutting flow andcontrol channels could alternatively be manipulated by electric ormagnetic fields, as described extensively above.

10. Additional Applications

The following represent further aspects of the present invention:present valves and pumps can be used for drug delivery (for example, inan implantable drug delivery device); and for sampling of biologicalfluids (for example, by storing samples sequentially in a column withplugs of spacer fluid therebetween, wherein the samples can be shuntedinto different storage reservoirs, or passed directly to appropriatesensor(s). Such a fluid sampling device could also be implanted in thepatient's body.

The present systems can also be used for devices which relieveover-pressure in vivo using a micro-valve or pump. For example, animplantable bio-compatible micro-valve can be used to relieveover-pressures in the eye which result from glaucoma. Other contemplateduses of the present switchable micro-valves include implantation in thespermatic duct or fallopian tube allowing reversible long-term orshort-term birth control without the use of drugs.

Further uses of the present invention include DNA sequencing whereby theDNA to be sequenced is provided with a polymerase and a primer, and isthen exposed to one type of DNA base (A, C, T, or G) at a time in orderto rapidly assay for base incorporation. In such a system, the basesmust be flowed into the system and excess bases washed away rapidly.Pressure driven flow, gated by elastomeric micro-valves in accordancewith the present invention would be ideally suited to allow for suchrapid flow and washing of reagents.

Other contemplated uses of the present micro-valve and micro-pumpsystems include uses with DNA chips. For example, a sample can be flowedinto a looped channel and pumped around the loop with a peristalticaction such that the sample can make many passes over the probes of theDNA array. Such a device would give the sample that would normally bewasted sitting over the non-complimentary probes the chance to bind to acomplimentary probe instead. An advantage of such a looped-flow systemis that it would reduce the necessary sample volume, and therebyincrease assay sensitivity.

Further applications exist in high throughput screening in whichapplications could benefit by the dispensing of small volumes of liquid,or by bead-based assays wherein ultrasensitive detection wouldsubstantially improve assay sensitivity.

Another contemplated application is the deposition of array of variouschemicals, especially oligonucleotides, which may or may not have beenchemically fabricated in a previous action of the device beforedeposition in a pattern or array on a substrate via contact printingthrough fluid channel outlets in the elastomeric device in closeproximity to a desired substrate, or by a process analogous to ink-jetprinting.

The present microfabricated elastomeric valves and pumps could also beused to construct systems for reagent dispensing, mixing and reactionfor synthesis of oligonucleotides, peptides or other biopolymers.

Further applications for the present invention include ink jet printerheads, in which small apertures are used to generate a pressure pulsesufficient to expel a droplet. An appropriately actuated micro-valve inaccordance with the present invention can create such a pressure pulse.The present micro-valves and pumps can also be used to digitallydispense ink or pigment, in amounts not necessarily as small as singledroplets. The droplet would be brought into contact with the mediumbeing printed on rather than be required to be fired through the air.

Yet further uses of the present invention would take advantage of theready removal and reattachment of the structure from an underlyingsubstrate such as glass, utilizing a glass substrate patterned with abinding or other material. This allows separate construction of apatterned substrate and elastomer structure. For instance, a glasssubstrate could be patterned with a DNA microarray, and an elastomervalve and pump structure sealed over the array in a subsequent step.

11. Additional Aspects of the Invention

The following represent further aspects of the present invention: theuse of a deflectable membrane to control flow of a fluid in amicrofabricated channel of an elastomeric structure; the use ofelastomeric layers to make a microfabricated elastomeric devicecontaining a microfabricated movable portion; and the use of anelastomeric material to make a microfabricated valve or pump.

A microfabricated elastomeric structure in accordance with oneembodiment of the present invention comprises an elastomeric blockformed with microfabricated recesses therein, a portion of theelastomeric block deflectable when the portion is actuated. The recessescomprise a first microfabricated channel and a first microfabricatedrecess, and the portion comprises an elastomeric membrane deflectableinto the first microfabricated channel when the membrane is actuated.The recesses have a width in the range of 10 μm to 200 μm and theportion has a thickness of between about 2 μm and 50 μm. Themicrofabricated elastomeric structure may be actuated at a speed of 100Hz or greater and contains substantially no dead volume when the portionis actuated.

A method of actuating an elastomeric structure comprises providing anelastomeric block formed with first and second microfabricated recessestherein, the first and second microfabricated recesses separated by amembrane portion of the elastomeric block deflectable into one of thefirst and second recesses in response to an actuation force, andapplying an actuation force to the membrane portion such that themembrane portion is deflected into one of the first and the secondrecesses.

A method of microfabricating an elastomeric structure in accordance withone embodiment of the present invention comprises forming a firstelastomeric layer on a substrate, curing the first elastomeric layer,and patterning a first sacrificial layer over the first elastomericlayer. A second elastomeric layer is formed over the first elastomericlayer, thereby encapsulating the first patterned sacrificial layerbetween the first and second elastomeric layers, the second elastomericlayer is cured, and the first patterned sacrificial layer is removedselective to the first elastomeric layer and the second elastomericlayer, thereby forming at least one first recess between the first andsecond layers of elastomer.

An alternative embodiment of a method of fabricating further comprisespatterning a second sacrificial layer over the substrate prior toforming the first elastomeric layer, such that the second patternedsacrificial layer is removed during removal of the first patternedsacrificial layer to form at least one recess along a bottom of thefirst elastomeric layer.

A microfabricated elastomeric structure in accordance with oneembodiment of the present invention comprises an elastomeric block, afirst channel and a second channel separated by a separating portion ofthe elastomeric structure, and a microfabricated recess in theelastomeric block adjacent to the separating portion such that theseparating portion may be actuated to deflect into the microfabricatedrecess. 66. Deflection of the separating portion opens a passagewaybetween the first and second channels.

A method of controlling fluid or gas flow through an elastomericstructure comprises providing an elastomeric block, the elastomericblock having first, second, and third microfabricated recesses, and theelastomeric block having a first microfabricated channel passingtherethrough, the first, second and third microfabricated recessesseparated from the first channel by respective first, second and thirdmembranes deflectable into the first channel, and deflecting the first,second and third membranes into the first channel in a repeatingsequence to peristaltically pump a flow of fluid through the firstchannel.

A method of microfabricating an elastomeric structure comprisesmicrofabricating a first elastomeric layer, microfabricating a secondelastomeric layer; positioning the second elastomeric layer on top ofthe first elastomeric layer; and bonding a bottom surface of the secondelastomeric layer onto a top surface of the first elastomeric layer.

12. Alternative Device Fabrication Methods

FIGS. 1-7A and 8-18 above show embodiments of multilayer softlithography and encapsulation methods, respectively, for fabricatingelastomer structures in accordance with the present invention. However,these fabrication methods are merely exemplary, and variations on thesetechniques may be employed to create elastomer structures.

For example, FIGS. 3 and 4 show fabrication of an elastomer structureutilizing multilayer soft lithography techniques, wherein therecess-bearing face of the upper elastomer layer is placed on top of thenon-recess-bearing face of the lower elastomer layer. However, thepresent invention is not limited to this configuration.

FIG. 50 shows elastomeric structure 5410 featuring an alternativeorientation of elastomeric layers, wherein recess-bearing faces 5400 and5402 of first and second elastomer layers 5404 and 5406 are respectivelyplaced into contact to create a larger-sized channel 5408.Alternatively, FIG. 51 shows an orientation of elastomeric layerswherein non-recess bearing faces 5500 and 5502 are placed into contact,such that recesses are present on opposite sides of the structure. Suchan elastomer structure 5512 could be sandwiched between substrates 5508and 5510 to produce channels 5504 and 5506 that cross-over each other.

FIGS. 52A-52D show various views of steps for constructing a fluidchannel bridging structure utilizing encapsulation methods.Specifically, FIG. 52A shows a cross-sectional view of the formation oflower elastomeric portion 5600 of bridging structure 5602. FIG. 52Bshows a cross-sectional view of the formation of upper elastomericportion 5604 of the bridging structure. FIG. 52C shows assembly of upperand lower elastomeric portions 5604 and 5600 to form bridging structure5602, wherein fluid flowing in channel 5606 bridges over cross-channel5608 through vias 5610 and 5612 and bridge portion 5614 as shown by thearrows. FIG. 52D shows a plan view of bridging structure 5602, withbridge portion 5614 of the upper elastomeric is shown in solid, and thefeatures in the underlying lower elastomer layer are shown in outline.This structure requires the formation of vias in the lower layer, butallows multiple liquid streams to flow in a single layer and cross overone another without mixing.

13. Composite Structures

As discussed above in conjunction with the micromirror array structureof FIG. 38, the fabricated elastomeric structures of the presentinvention may be combined with nonelastomeric materials to createcomposite structures. Fabrication of such composite structures is nowdiscussed in further detail.

FIG. 53A shows a cross-sectional view of one embodiment of a compositestructure in accordance with the present invention. FIG. 53A showscomposite valve structure 5700 including first, thin elastomer layer5702 overlying semiconductor-type substrate 5704 having channel 5706formed therein. Second, thicker elastomer layer 5708 overlies firstelastomer layer 5702. Actuation of first elastomer layer 5702 to driveit into channel 5706, will cause composite structure 5700 to operate asa valve.

FIG. 53B shows a cross-sectional view of a variation on this theme,wherein thin elastomer layer 5802 is sandwiched between two hard,semiconductor substrates 5804 and 5806, with lower substrate 5804featuring channel 5808. Again, actuation of thin elastomer layer 5802 todrive it into channel 5808 will cause composite structure 5810 tooperate as a valve.

The structures shown in FIG. 53 or 53B may be fabricated utilizingeither the multilayer soft lithography or encapsulation techniquesdescribed above. In the multilayer soft lithography method, theelastomer layer(s) would be formed and then placed over thesemiconductor substrate bearing the channel. In the encapsulationmethod, the channel would be first formed in the semiconductorsubstrate, and then the channel would be filled with a sacrificialmaterial such as photoresist. The elastomer would then be formed inplace over the substrate, with removal of the sacrificial materialproducing the channel overlaid by the elastomer membrane. As isdiscussed in detail below in connection with bonding of elastomer toother types of materials, the encapsulation approach may result in astronger seal between the elastomer membrane component and theunderlying nonelastomer substrate component.

As shown in FIGS. 53 and 53B, a composite structure in accordance withembodiments of the present invention may include a hard substrate thatbears a passive feature such as a channels. However, the presentinvention is not limited to this approach, and the underlying hardsubstrate may bear active features that interact with an elastomercomponent bearing a recess. This is shown in FIG. 55, wherein compositestructure 5900 includes elastomer component 5902 containing recess 5904having walls 5906 and ceiling 5908. Ceiling 5908 forms flexible membraneportion 5909. Elastomer component 5902 is sealed against substantiallyplanar nonelastomeric component 5910 that includes active device 5912.Active device 5912 may interact with material present in recess 5904and/or flexible membrane portion 5909.

In the micromirror array embodiment previously described in conjunctionwith FIG. 38, the underlying substrate contained active structures inthe form of an electrode array. However, many other types of activestructures may be present in the nonelastomer substrate. Activestructures that could be present in an underlying hard substrateinclude, but are not limited to, resistors, capacitors, photodiodes,transistors, chemical field effect transistors (chem FET's),amperometric/coulometric electrochemical sensors, fiber optics, fiberoptic interconnects, light emitting diodes, laser diodes, verticalcavity surface emitting lasers (VCSEL's), micromirrors, accelerometers,pressure sensors, flow sensors, CMOS imaging arrays, CCD cameras,electronic logic, microprocessors, thermistors, Peltier coolers,waveguides, resistive heaters, chemical sensors, strain gauges,inductors, actuators (including electrostatic, magnetic,electromagnetic, bimetallic, piezoelectric, shape-memory-alloy based,and others), coils, magnets, electromagnets, magnetic sensors (such asthose used in hard drives, superconducting quantum interference devices(SQUIDS) and other types), radio frequency sources and receivers,microwave frequency sources and receivers, sources and receivers forother regions of the electromagnetic spectrum, radioactive particlecounters, and electrometers.

As is well known in the art, a vast variety of technologies can beutilized to fabricate active features in semiconductor and other typesof hard substrates, including but not limited printed circuit board(PCB) technology, CMOS, surface micromachining, bulk micromachining,printable polymer electronics, and TFT and otheramorphous/polycrystalline techniques as are employed to fabricate laptopand flat screen displays.

A composite structure in accordance with one embodiment of the presentinvention comprises a nonelastomer substrate having a surface bearing afirst recess, a flexible elastomer membrane overlying the non-elastomersubstrate, the membrane able to be actuated into the first recess, and alayer overlying the flexible elastomer membrane.

An embodiment of a method of forming a composite structure comprisesforming a recess in a first nonelastomer substrate, filling the recesswith a sacrificial material, forming a thin coat of elastomer materialover the nonelastomer substrate and the filled recess, curing theelastomer to form a thin membrane, and removing the sacrificialmaterial.

A composite structure in accordance with an alternative embodiment ofthe present invention comprises an elastomer component defining a recesshaving walls and a ceiling, the ceiling of the recess forming a flexiblemembrane portion, and a substantially planar nonelastomer componentsealed against the elastomer component, the nonelastomer componentincluding an active device interacting with at least one of the membraneportion and a material present in the recess.

A method of fabricating a composite structure comprising forming arecess in an elastomer component, the recess including walls and aceiling, the ceiling forming a flexible membrane portion, positioning amaterial within the recess, forming a substantially planar nonelastomercomponent including an active device, and sealing the elastomercomponent against the nonelastomer component, such that the activedevice may interact with at least one of the membrane portion and thematerial.

A variety of approaches can be employed to seal the elastomericstructure against the nonelastomeric substrate, ranging from thecreation of a Van der Waals bond between the elastomeric andnonelastomeric components, to creation of covalent or ionic bondsbetween the elastomeric and nonelastomeric components of the compositestructure. Example approaches to sealing the components together arediscussed below, approximately in order of increasing strength.

A first approach is to rely upon the simple hermetic seal resulting fromVan der Waals bonds formed when a substantially planar elastomer layeris placed into contact with a substantially planar layer of a harder,non-elastomer material. In one embodiment, bonding of RTV elastomer to aglass substrate created a composite structure capable of withstanding upto about 3-4 psi of pressure. This may be sufficient for many potentialapplications.

A second approach is to utilize a liquid layer to assist in bonding. Oneexample of this involves bonding elastomer to a hard glass substrate,wherein a weakly acidic solution (5 μl HCl in H₂O, pH 2) was applied toa glass substrate. The elastomer component was then placed into contactwith the glass substrate, and the composite structure baked at 37° C. toremove the water. This resulted in a bond between elastomer andnon-elastomer able to withstand a pressure of about 20 psi. In thiscase, the acid may neutralize silanol groups present on the glasssurface, permitting the elastomer and non-elastomer to enter into goodVan der Waals contact with each other.

Exposure to ethanol can also cause device components to adhere together.In one embodiment, an RTV elastomer material and a glass substrate werewashed with ethanol and then dried under Nitrogen. The RTV elastomer wasthen placed into contact with the glass and the combination baked for 3hours at 80° C. Optionally, the RTV may also be exposed to a vacuum toremove any air bubbles trapped between the slide and the RTV. Thestrength of the adhesion between elastomer and glass using this methodhas withstood pressures in excess of 35 psi. The adhesion created usingthis method is not permanent, and the elastomer may be peeled off of theglass, washed, and resealed against the glass. This ethanol washingapproach can also be employed used to cause successive layers ofelastomer to bond together with sufficient strength to resist a pressureof 30 psi. In alternative embodiments, chemicals such as other alcoholsor diols could be used to promote adhesion between layers.

An embodiment of a method of promoting adhesion between layers of amicrofabricated structure in accordance with the present inventioncomprises exposing a surface of a first component layer to a chemical,exposing a surface of a second component layer to the chemical, andplacing the surface of the first component layer into contact with thesurface of the second elastomer layer.

A third approach is to create a covalent chemical bond between theelastomer component and functional groups introduced onto the surface ofa nonelastomer component. Examples of derivitization of a nonelastomersubstrate surface to produce such functional groups include exposing aglass substrate to agents such as vinyl silane or aminopropyltriethoxysilane (APTES), which may be useful to allow bonding of the glass tosilicone elastomer and polyurethane elastomer materials, respectively.

A fourth approach is to create a covalent chemical bond between theelastomer component and a functional group native to the surface of thenonelastomer component. For example, RTV elastomer can be created withan excess of vinyl groups on its surface. These vinyl groups can becaused to react with corresponding functional groups present on theexterior of a hard substrate material, for example the Si—H bondsprevalent on the surface of a single crystal silicon substrate afterremoval of native oxide by etching. In this example, the strength of thebond created between the elastomer component and the nonelastomercomponent has been observed to exceed the materials strength of theelastomer components.

The discussion of composite structures has so far focused upon bondingan elastomer layer to an underlying non-elastomer substrate. However,this is not required by the present invention.

As previously described, a microfabricated elastomer structure may besliced vertically, often preferably along a channel cross section. Inaccordance with embodiments of the present invention, a non-elastomercomponent may be inserted into the elastomer structure that has beenopened by such a cut, with the elastomer structure then resealed. Oneexample of such an approach is shown in FIGS. 57A-57C, which illustratescross-sectional views of a process for forming a flow channel having amembrane positioned therein. Specifically, FIG. 57A shows across-section of a portion of device 6200 including elastomer membrane6202 overlying flow channel 6204, and elastomer substrate 6206.

FIG. 57B shows the results of cutting device 6200 along vertical line6208 extending along the length of flow channel 6204, such that halves6200 a and 6200 b are formed. FIG. 57C shows insertion of permeablemembrane element 6210 between halves 6200 a and 6200 b, followed byattachment of halves 6200 a and 6200 b to permeable membrane 6210. As aresult of this configuration, the flow channel of the device actuallycomprises channel portions 6204 a and 6204 b separated by permeablemembrane 6210.

The structure of FIG. 57C could be utilized in a variety ofapplications. For example, the membrane could be used to performdialysis, altering the salt concentration of samples in the flowchannel. The membrane of the structure of FIG. 57C could also be used topurify samples. Yet another potential application for the structure ofFIG. 57C is to isolate the products of a reaction, for example to removea product from an enzymatic reaction in order to avoid productinhibition. Depending upon the composition of the membrane, an organicsolvent may be present on one side of the membrane while an aqueousbuffer is present on the other side of the membrane. Yet anotherpossible function of the membrane would be to bind particular chemicalmoieties such as proteins, peptides or DNA fragments for purification,catalysis or analysis.

An embodiment of a method of fabricating an elastomeric structurecomprises cutting the first elastomer structure along a vertical sectionto form a first elastomer portion and a second elastomer portion; andbonding the first elastomer structure to another component.

14. Priming of Flow Channels

One potentially useful property of elastomeric structures is theirpermeability to gases.

Specifically, elastomer materials may permit diffusion of certain gasspecies, while preventing diffusion of liquids. This characteristic maybe very useful in manipulating small volumes of fluid.

For example, fluidic devices having complex channel architectures ingeneral must address issues of priming the channels. As fluid isinitially injected into a complicated channel structure, it follows thepath of least resistance and may leave some regions of the structureunfilled. It is generally difficult or impossible to fill dead-endregions of the structure with liquid.

Accordingly, one approach of the present invention exploits gaspermeability of certain elastomer materials to fill channel structuresincluding dead end channels and chambers. In accordance with oneembodiment of a method for priming a microfluidic structure, initiallyall but one of the channel entries into the structure are plugged. Aneutral buffer is then injected into the remaining channel andmaintained under pressure. The pressurized buffer fills the channels,compressing in front of it any gas present in the channel. Thuscompressed, gas trapped in the channel is forced to diffuse out of thestructure through the elastomer.

In this manner, a liquid sample may be introduced into the flow channelsof a microfabricated device without producing unwanted pockets of gas.By injecting a liquid sample under pressure into the flow channels of amicrofluidic device and then maintaining the injection pressure for agiven time, the liquid sample fills the channel and any gas resultingpockets within the channel diffuse out of the elastomer material.According to this method, the entire microfluidic structure may rapidlybe filled from a single via. Dead end chambers filled with liquidutilizing this method may be employed in storage, metering, and mixingapplications.

The gas permeability of elastomer materials may result in theintroduction of gases into flow channels where pneumatic (air-driven)actuation systems are employed. Such pneumatic valves may be operated athigh control pressures in order to withstand high back pressures. Undersuch operating conditions, gas may diffuse across a relatively thinmembrane, thereby introducing bubbles into the flow channel. Suchunwanted diffusion of air through the membrane can be avoided byutilizing a control medium to which the elastomer is much lesspermeable, i.e. a gas which diffuses much more slowly through theelastomer or a liquid. As described above, control lines function wellto transmit a control pressure when filled with water or oil, and do notintroduce bubbles into the flow channels. Furthermore, using the primingmethod described above, it is possible to fill dead end control channelswith fluid and hence no modification of an existing air-driven controlstructure would be required.

An embodiment of a method of filling a microfabricated elastomericstructure with fluid in accordance with the present invention comprisesproviding an elastomer block having a flow channel, the elastomer blockcomprising an elastomer material known to be permeable to a gas. Theflow channel is filled with the gas, fluid is injected under pressureinto the flow channel, and gas remaining in the flow channel ispermitted to diffuse out of the elastomer material.

15. Metering by Volume Exclusion

Many high throughput screening and diagnostic applications call foraccurate combination and of different reagents in a reaction chamber.Given that it is frequently necessary to prime the channels of amicrofluidic device in order to ensure fluid flow, it may be difficultto ensure mixed solutions do not become diluted or contaminated by thecontents of the reaction chamber prior to sample introduction.

Volume exclusion is one technique enabling precise metering of theintroduction of fluids into a reaction chamber. In this approach, areaction chamber may be completely or partially emptied prior to sampleinjection. This method reduces contamination from residual contents ofthe chamber contents, and may be used to accurately meter theintroduction of solutions in a reaction chamber.

Specifically, FIGS. 58A-58D show cross-sectional views of a reactionchamber in which volume exclusion is employed to meter reactants. FIG.58A shows a cross-sectional view of portion 6300 of a microfluidicdevice comprising first elastomer layer 6302 overlying second elastomerlayer 6304. First elastomer layer 6302 includes control chamber 6306 influid communication with a control channel (not shown). Control chamber6306 overlies and is separated from dead-end reaction chamber 6308 ofsecond elastomer layer 6304 by membrane 6310. Second elastomer layer6304 further comprises flow channel 6312 leading to dead-end reactionchamber 6308. As previously described, first reactant X may beintroduced under pressure into dead-end reaction chamber 6308.

FIG. 58B shows the result of a pressure increase within control chamber6306.

Specifically, increased control chamber pressure causes membrane 6310 toflex downward into reaction chamber 6308, reducing by volume V theeffective volume of reaction chamber 6308. This in turn excludes anequivalent volume V of reactant from reaction chamber 6308, such thatvolume V of first reactant Xis output from flow channel 6312. The exactcorrelation between a pressure increase in control chamber 6306 and thevolume of material output from flow channel 6312 can be preciselycalibrated.

As shown in FIG. 58C, while elevated pressure is maintained withincontrol chamber 6306, volume V′ of second reactant Y is placed intocontact with flow channel 6312 and reaction chamber 6308.

In the next step shown in FIG. 58D, pressure within control chamber 6306is reduced to original levels. As a result, membrane 6310 relaxes andthe effective volume of reaction chamber 6308 increases. Volume V ofsecond reactant Y is sucked into the device. By varying the relativesize of the reaction and control chambers, it is possible to accuratelymix solutions at a specified relative concentration. It is worth notingthat the amount of the second reactant Y that is sucked into the deviceis solely dependent upon the excluded volume V, and is independent ofvolume V′ of Y made available at the opening of the flow channel.

While FIGS. 58A-58D show a simple embodiment of the present inventioninvolving a single reaction chamber, in more complex embodimentsparallel structures of hundreds or thousands of reaction chambers couldbe actuated by a pressure increase in a single control line.

Moreover, while the above description illustrates two reactants beingcombined at a relative concentration that fixed by the size of thecontrol and reaction chambers, a volume exclusion technique could beemployed to combine several reagents at variable concentrations in asingle reaction chamber. One possible approach is to use several,separately addressable control chambers above each reaction chamber. Anexample of this architecture would be to have ten separate control linesinstead of a single control chamber, allowing ten equivalent volumes tobe pushed out or sucked in.

Another possible approach would utilize a single control chamberoverlying the entire reaction chamber, with the effective volume of thereaction chamber modulated by varying the control chamber pressure. Inthis manner, analog control over the effective volume of the reactionchamber is possible. Analog volume control would in turn permit thecombination of many solutions reactants at arbitrary relativeconcentrations.

An embodiment of a method of metering a volume of fluid in accordancewith the present invention comprises providing a chamber having a volumein an elastomeric block separated from a control recess by anelastomeric membrane, and supplying a pressure to the control recesssuch that the membrane is deflected into the chamber and the volume isreduced by a calibrated amount, thereby excluding from the chamber thecalibrated volume of fluid.

16. Protein Crystallization

As just described, embodiments of microfabricated elastomeric devices inaccordance with the present invention permit extremely precise meteringof fluids for mixing and reaction purposes. However, precise controlover metering of fluid volumes can also be employed to promotecrystallization of molecules such as proteins.

Protein recrystallization is an important technique utilized to identifythe structure and function of proteins. Recrystallization is typicallyperformed by dissolving the protein in an aqueous solution, and thendeliberately adding a countersolvent to alter the polarity of thesolution and thereby force the protein out of solution and into thesolid phase. Forming a high quality protein crystal is generallydifficult and sometimes impossible, requiring much trial and error withthe identities and concentrations of both the solvents and the countersolvents employed.

Accordingly, FIG. 67 shows a plan view of protein crystallization systemthat allows mass recrystallization attempts. Protein crystallizationsystem 7200 comprises control channel 7202 and flow channels 7204 a,7204 b, 7204 c, and 7204 d. Each of flow channels 7204 a, 7204 b, 7204c, and 7204 d feature dead-end chambers 7206 that serve as the site forrecrystallization. Control channel 7202 features a network of controlchambers 7205 of varying widths that overlie and are separated fromchambers 7206 by membranes 7208 having the same widths as controlchambers 7205. Although not shown to clarify the drawing, a secondcontrol featuring a second network of membranes may be utilized tocreate stop valves for selectively opening and closing the openings todead-end chambers 7206. A full discussion of the function and role ofsuch stop valves is provided below in conjunction with FIG. 70.

Operation of protein crystallization system 7200 is as follows.Initially, an aqueous solution containing the target protein is flushedthrough each of flow channels 7204 a, 7204 b, 7204 c, and 7204 d,filling each dead-end chamber 7206. Next, a high pressure is applied tocontrol channel 7202 to deflect membranes 7208 into the underlyingchambers 7206, excluding a given volume from chamber 7206 and flushingthis excluded volume of the original protein solution out of chamber7206.

Next, while pressure is maintained in control channel 7202, a differentcountersolvent is flowed into each flow channel 7204 a, 7204 b, 7204 c,and 7204 d. Pressure is then released in control line 7202, andmembranes 7208 relax back into their original position, permitting theformerly excluded volume of countersolvent to enter chambers 7206 andmix with the original protein solution. Because of the differing widthsof control chambers 7205 and underlying membranes 7208, a variety ofvolumes of the countersolvent enters into chambers 7206 during thisprocess.

For example, chambers 7206 a in the first two rows of system 7200 do notreceive any countersolvent because no volume is excluded by an overlyingmembrane. Chambers 7106 b in the second two rows of system 7200 receivea volume of countersolvent that is 1:5 with the original proteinsolution. Chambers 7206 c in the third two rows of system 7200 receive avolume of countersolvent that is 1:3 with the original protein solution.Chambers 7206 d in the fourth two rows of system 7200 receive a volumeof countersolvent that is 1:2 with the original protein solution, andchambers 7206 e in the fifth two rows of system 7200 receive a volume ofcountersolvent that is 4:5 with the original protein solution.

Once the countersolvent has been introduced into the chambers 7206, theymay be resealed against the environment by again applying a highpressure to control line 7202 to deflect the membranes into thechambers. Resealing may be necessary given that recrystallization canrequire on the order of days or weeks to occur. Where visual inspectionof a chamber reveals the presence of a high quality crystal, the crystalmay be physically removed from the chamber of the disposable elastomersystem.

While the above description has described a protein crystallizationsystem that relies upon volume exclusion to meter varying amounts ofcountersolvent, the invention is not limited to this particularembodiment. Accordingly, FIG. 70 shows a plan view of proteincrystallization system wherein metering of different volumes ofcountersolvent is determined by photolithography during formation of theflow channels.

Protein crystallization system 7500 comprises flow channels 7504 a, 7504b, 7504 c, and 7504 d. Each of flow channels 7504 a, 7504 b, 7504 c, and7504 d feature dead-end chambers 7506 that serve as the site forrecrystallization.

System 7500 further comprises two sets of control channels. First set7502 of control channels overlie the opening of chambers 7506 and definestop valves 7503 that, when actuated, block access to chambers 7506.Second control channels 7505 overlie flow channels 7504 a-d and definesegment valves 7507 that, when actuated, block flow between differentsegments 7514 of a flow channel 7404.

Operation of protein crystallization system 7500 is as follows.Initially, an aqueous solution containing the target protein is flushedthrough each of flow channels 7504 a, 7504 b, 7504 c, and 7504 d,filling dead-end chambers 7506. Next, a high pressure is applied tocontrol channel 7502 to actuate stop valves 7503, thereby preventingfluid from entering or exiting chambers 7506.

While maintaining stop valves 7503 closed, each flow channel 7504 a-d isthen filled with a different countersolvent. Next, second control line7505 is pressurized, isolating flow channels 7504 a-d into segments 7514and trapping differing volumes of countersolvent. Specifically, as shownin FIG. 70 segments 7514 are of unequal volumes. During formation ofprotein crystallization structure 7500 by soft lithography,photolithographic techniques are employed to define flow channels 7504a-d having segments 7514 of different widths 7514 a and lengths 7514 b.

Thus, when pressure is released from first control line 7502 and stopvalves 7503 open, a different volume of countersolvent from the varioussegments 7514 may diffuse into chambers 7506. In this manner, precisedimensions defined by photolithography can be employed to determine thevolume of countersolvent trapped in the flow channel segments and thenintroduced to the protein solution. This volume of countersolvent inturn establishes the environment for crystallization of the protein.

A protein crystallization system in accordance with one embodiment ofthe present invention comprises an elastomeric block including amicrofabricated chamber having a volume and receiving a proteinsolution, and a microfabricated flow channel in fluid communication withthe chamber, the flow channel receiving a countersolvent and introducinga fixed volume of countersolvent to the chamber.

17. Pressure Amplifier

As discussed above, certain embodiments of microfabricated elastomerdevices according to the present invention utilize pressures to controlthe flow of fluids. Accordingly, it may be useful to include within thedevice structures that enable the amplification of applied pressures inorder to enhance these effects.

FIGS. 59A and 59B show cross-sectional and plan views respectively, ofan embodiment of a linear amplifier constructed utilizingmicrofabrication techniques in accordance with the present invention.Pressure amplifier 6400 comprises first (control) elastomer layer 6402overlying second (amplifying) elastomer layer 6404 that in turn overliesthird (flow) elastomer layer 6406. Third elastomer layer 6406 in turnoverlies substrate 6409.

Third elastomer layer 6406 encloses flow channel 6408 separated fromoverlying amplifying elastomer layer 6404 by membrane 6410. Pyramidalamplifying element 6412 includes lower surface 6414 in contact withmembrane 6410 and upper surface 6416 in contact with first elastomerlayer 6402. Area A₁ of upper surface 6416 of pyramidal amplifyingelement 6412 is larger than area A₂ of lower surface 6414.

As a result of application of a pressure p₁ in control channel 6415 offirst elastomer layer 6402, a force F₁ is communicated to upper surface6416 of pyramid structure 6412. Pyramid 6412 in turn transfers force F₁to lower surface 6414 having a smaller area. Since the force is equal topressure multiplied by area (F=pA), and since the force applied to thetop of pyramid structure 6412 is equal to the force exerted by the baseof pyramid structure 6412, p₁A₁=p₂A₂. Since A₁>A₂, membrane 6410 willexperience an amplified pressure p₂, p₂>P₁. The resulting amplifyingfactor can be estimated by simply comparing the two areas A_(l) and A₂:

p ₂ /{dot over (p)} ₁ ˜A ₁ /A ₂,  (2)

where

-   -   p₂/p₁=pressure amplifying factor.

Equation (2) is an approximation due to the elasticity of the amplifierstructure itself, which will generally act to reduce the amplifyingeffect.

An example of a method for fabricating a pressure amplifier structure isnow provided. A first, control elastomer layer featuring a controlchannel of width 200 μm and height 10 μm is fabricated utilizing 5A:1BPDMS over a mold. A second, flow elastomer layer featuring a roundedflow channel of width 50 μm and height 10 μm is similarly fabricated byspinning 5A:1B PDMS on a mold at 4000 rpm for 60 sec, generating amembrane having a thickness of 15

The third, amplifying elastomer layer is formed by providing a 3″silicon wafer of lattice type <100> bearing an oxide layer of thickness20,000 Å oxide layer (Silicon Quest International). A pattern ofphotoresist 5740 (Shipley Microelectronics) reflecting the dimension ofthe upper surface (in this case a square having sides 200 μm) of thepyramidal amplification structure is then formed. Using the patternedphotoresist as a mask, oxide of the wafer exposed by the photoresistpattern is etched with HF.

Next, the patterned oxide layer is utilized as a mask for the removal ofexposed underlying silicon to form a mold for the pyramidal amplifyingstructure. Specifically, exposed silicon on the wafer is wet etched at80° C. with 30% KOH (w/v), resulting in formation of a trench having awalls inclined at an angle of 54.7°. The etch rate of silicon utilizingthis chemistry is 1 μm/min, allowing precise control over the depth ofthe trench and hence the height of the amplifying structure molded andthe resulting amplification factor. In this example, etching for 20 minresulting in a 20 μm deep etch was employed.

After etching of the silicon is completed, the photoresist is removedwith acetone and the oxide layer is etched away with HF. Afterward, thesilicon mold is treated with Trimethylchlorosilane (TMCS, Sigma) and20A:1B RTV is spun at 2000 rpm for 60 sec onto the silicon moldincluding the etched trench, and then baked for 60 min at 80° C. Next,the control layer is aligned over the amplifying layer. The two layersare baked together for an additional hour. The two layers are peeled ofcarefully from the (amplifier) mold and are aligned on the flow channel.After an additional one hour baking the complete device is removed fromthe mold.

FIG. 59C shows a photograph of the finally assembled device in the openstate. FIG. 59D shows a photograph of the finally assembled device inthe closed state in response to application of a pressure of 18 psipressure to control channel 6415, resulting in an amplification factorof approximately 1.3.

An embodiment of a pressure amplifier in accordance with the presentinvention comprises an elastomeric block formed with first and secondmicrofabricated recesses therein, and an amplifier structure having afirst surface area in contact with the first recess and a second surfacearea in contact with the second recess. The first surface area is largerthan the second surface area such that a pressure in the first recess iscommunicated by the amplifier structure as an amplified pressure to thesecond recess.

A method for amplifying a pressure in a flow channel of amicrofabricated elastomer structure comprises providing an elastomerblock including a first recess in contact with a first area of anamplifier structure and second recesses in contact with a second area ofthe amplifier structure, applying a pressure to the first area; andcommunicating the pressure to the second area, the second area smallerthan the first area such that the pressure communicated to the secondrecess is amplified.

18. Check Valve

One potentially useful structure in conventional fluid handling devicesis a check valve which permits the flow of fluid in only one directionthrough a conduit. FIGS. 61A and 61B show plan and cross-sectionalviews, respectively, of a microfabricated one-way valve structure inaccordance with an embodiment of the present invention. Microfabricatedstructure 6600 includes flow channel 6602 formed in elastomer 6604. Leftportion 6602 a and right portion 6602 b of flow channel 6602 are influid communication with each other through one-way valve 6606.

Specifically, microfluidic one-way valve 6606 permits fluid to flow indirection through flow channel 6602 and moveable flap 6608, but not inopposite direction Flap 6608 is integral with ceiling 6602 c of flowchannel 6602 and does not contact bottom 6602 d or sides 6602 e of flowchannel 6602, thus allowing flap 6608 to swing freely. Flap 6608 is ableto seal against left portion 6602 a of flow channel 6602 because it isboth larger in width and in height than the cross-section of the rightchannel portion. Alternatively, the walls of the right channel mayfeature protuberances that inhibit movement of the flap.

Valve 6606 operates passively, relying upon channel geometry and theproperties of the elastomer rather than upon external forces.Specifically, as fluid flows from left to right in direction M→, flap6608 is able to swing open into right hand side 6602 b of flow channel.However, when the direction of fluid flow reverses to flap 6608 sealsagainst the opening of left hand side 6602 a of flow channel 6602.

FIGS. 62A-62E show cross-sectional views of a process for fabricating aone way valve in accordance with the present invention. In FIG. 62A,first micomachined silicon mold 6620 is formed using first patternedphotoresist layer 6622 defining first recess 6624 that will in turndefine bottom-sealing portion 6626 of the flow channel. In FIG. 62B,elastomer is poured onto mold 6620 and solidified, such that removal offirst molded piece 6625 produces elevated bottom-sealing portion 6626.In FIG. 62C, second micromachined silicon mold 6630 is formed usingsecond patterned photoresist layer 6632 defining second recess 6634 thatwill in turn define the ceiling and the flap of the flow channel. InFIG. 62D, elastomer is poured onto second mold 6630 and cured, such thatremoval of second molded piece 6636 includes projecting flap 6608. InFIG. 62E, first molded piece 6625 and second molded piece 6636 arebonded together to produce intervening flow channel 6602 and one-wayvalve 6606.

A one-way valve in accordance with an embodiment of the presentinvention comprises a microfabricated channel formed in an elastomericblock, a flap integral with the elastomeric block projecting into thechannel and blocking the channel, the flap deflectable to permit fluidto flow in only a first direction.

19. Fluidics

Fluidics refers to techniques which utilize flowing fluids as a signalbearing medium, and which exploit properties of fluid dynamics formodification (switching, amplification) of those signals. Fluidics isanalogous to electronics, wherein a flow of electrons is used as thesignal bearing medium and electro-magnetic properties are utilized forsignal switching and amplification.

Accordingly, another application of microfabricated structures of thepresent invention is in fluidics circuits. One embodiment of a fluidiclogic device in accordance with the present invention comprises anelastomeric block, and a plurality of microfabricated channels formed inthe elastomeric block, each channel containing a pressure or flowrepresenting a signal, a change in the pressure or flow in a firstchannel resulting in a change in the pressure or fluid flow in a secondchannel consistent with a logic operation.

Fluidic logic structures in accordance with embodiments of the presentinvention offer the advantage of compact size. Another advantage offluidic logic structures of the present invention is their potential usein radiation resistant applications. A further advantage of fluidiclogic circuits is that, being non-electronic, such fluidic logiccircuitry may not be probed by electro magnetic sensors, thus offering asecurity benefit.

Yet another advantage of fluidic logic circuits is that they can bereadily integrated into microfluidic systems, enabling “onboard” controland reducing or eliminating the need for an external control means. Forinstance, fluidic logic could be used to control the speed of aperistaltic pump based on the downstream pressure, thus allowing directpressure regulation without external means. Similar control using anexternal means would be much more complicated and difficult toimplement, requiring a separate pressure sensor, transduction of thepressure signal into an electrical signal, software to control the pumprate, and external hardware to transduce the control signal intopneumatic control.

One fundamental logic structure is a NOR gate. The truth table for a NORlogic structure is given below in Table 1:

TABLE 1 NOR Gate Truth Table INPUT 1 INPUT 2 OUTPUT high high low highlow low low high low low low high

One simple embodiment of a NOR gate structure fabricated in accordancewith the present invention comprises a flow channel including an inletportion and an outlet portion, at least two control channels adjacent tothe flow channel and separated from the first channel by respectivefirst and second elastomer membranes, such that application of pressuresto the control channels deflects at least one of the first and secondmembranes into the flow channel to reflect a pressure at the outletconsistent with a NOR-type truth table.

FIG. 63 shows a plan view of an alternative embodiment of a NOR logicstructure in accordance with the present invention that is fabricatedfrom pressure amplifier structures. NOR gate 6900 comprises input flowchannels 6902 and 6904 that contain a pressurized flow of fluid. Controlchannels 6906 and 6908 are orthogonal to and overlie flow channels 6902and 6904, forming valves 6910 a, 6910 b, 6910 c, and 6910 d. Afterpassing underneath control channels 6906 and 6908, flow channels 6902and 6904 merge to form a single output flow channel 6912. NOR gate 6900operates as follows.

Where an input pressure signal to each of control lines 6906 and 6908 islow, valves 6910 a-d are open and fluid flows freely through flowchannels 6902 and 6904, resulting in a high combined pressure at outputflow channel 6912. Where a pressure higher than that present in flowchannels 6902 and 6904 is introduced into first control line 6906,valves 6910 a and 6910 b both close, resulting in a low pressure atoutput flow channel 6912. Similarly, where a pressure higher than thatpresent in flow channels 6902 and 6904 is introduced into second controlline 6908, one way valves 6910 c and 6910 d close, and the output atoutput flow channel 6912 is also low. Of course, application of highpressures in both control lines 6906 and 6908 still results in a lowpressure at output 6912.

NOR gate 6900 can be linked with other logic devices to form morecomplex logic structures. For example, FIG. 63 shows the output of firstNOR gate 6900 serves as one of the inputs (control lines) for second NORgate 6950, which is likely formed in an elastomer layer underlying firstNOR gate 6900. It may be necessary to amplify the pressure output fromfirst NOR gate 6900 in order to achieve the necessary control.

Moreover, signal amplification in accordance with embodiments of thepresent invention can be utilized to maintain control pressure signalsand information-carrying (flow) pressures at approximately the samemagnitude, Such approximately parity between information and controlsignals is one hallmark of conventional digital electronics controlsystems

Absent the use of amplified control pressures, fluidics applications inaccordance with embodiments of the present invention would generallyrequire control pressures larger than the flow pressures, in order tocontrol the flows of fluids through the device. However, using pressureamplification in accordance with embodiments of the present invention,pressures in the control and flow channels of a fluidics circuit can beof approximately the same order of magnitude.

Another basic building block of logic structures such as AND and ORgates is the diode. Accordingly, FIG. 64 shows a plan view of oneembodiment of an AND gate structure fabricated utilizing fluidic checkvalves in accordance with the present invention as diodes. The truthtable for an AND logic structure is given below in Table 2:

TABLE 2 AND Gate Truth Table INPUT 1 INPUT 2 OUTPUT high high high highlow low low high low low low low

Operation in accordance with this truth table is possible utilizingstructure 7000 of FIG. 64. Specifically, AND gate structure 7000includes inlet portion 7002 a flow channel 7002 in fluid communicationwith outlet portion of flow channel 7002 b through flow resistor 7004.First and second control channels 7006 and 7008 are in communicationwith flow channel 7002 at junction 7002 c immediately upstream of flowresistor 7004 through first one way valve 7010 and second one way valve7012 respectively.

Only where first control channel 7006 and second control channel 7008are pressurized (each corresponding to a high logic state) will a highpressure flow emerge from flow channel output portion 7002 b. Where thepressure in first control channel 7006 is low, the pressure in secondcontrol channel 7008 is low, or the pressures in both first and secondcontrol channels 7006 and 7008 are low, fluid entering device 7000through inlet 7002 a will encounter back pressure from flow resistor7004 at junction 7002 c, and in response flow out through one or both ofone way valves 7010 and 7012 and respective control channels 7006 and7008. More complex logic structures can be created by connecting an ANDgate with other logic structures in various combinations.

Monolithic Microfabricated Valves and Pumps by Multilayer SoftLithography

Soft lithography is an alternative to silicon-based micromachining thatuses replica molding of nontraditional elastomeric materials tofabricate stamps and microfluidic channels. We describe here anextension to the soft lithography paradigm, multilayer soft lithography,with which devices consisting of multiple layers may be fabricated fromsoft materials. We used this technique to build active microfluidicsystems containing on-off valves, switching valves, and pumps entirelyout of elastomer. The softness of these materials allows the deviceareas to be reduced by more than two orders of magnitude compared withsilicon-based devices. The other advantages of soft lithography, such asrapid prototyping, ease of fabrication, and biocompatibility, areretained.

(A) Process flow for multilayer soft lithography. The elastomer usedhere is General Electric Silicones RTV 615. Part “A” contains apolydimethylsiloxane bearing vinyl groups and a platinum catalyst; part“B” contains a cross-linker containing silicon hydride (Si—H) groups,which form a covalent bond with vinyl groups. RTV 615 is normally usedat a ratio of 10 A:1 B. For bonding, one layer is made with 30 A:1 B(excess vinyl groups) and the other with 3 A:1 B (excess Si—H groups).The top layer is cast thick (˜4 mm) for mechanical stability, whereasthe other layers are cast thin. The thin layer was created byspin-coating the RTV mixture on a microfabricated mold at 2000 rpm for30 s, yielding a thickness of ˜40 Each layer was separately baked at 80°C. for 1.5 hours. The thick layer was then sealed on the thin layer, andthe two were bonded at 80° C. for 1.5 hours. Molds were patternedphotoresist on silicon wafers. Shipley SJR 5740 photoresist was spun at2000 rpm, patterned with a high-resolution transparency film as a mask,and developed to yield inverse channels of 10 μm in height. When bakedat 200° C. for 30 min, the photoresist reflows and the inverse channelsbecome rounded. Molds were treated with trimethylchlorosilane vapor for1 min before each use to prevent adhesion of silicone rubber. (B)Schematic of valve closing for square and rounded channels. The dottedlines indicate the contour of the top of the channel for rectangular(left) and rounded (right) channels as pressure is increased. Valvesealing can be inspected by observing the elastomer-substrate interfaceunder an optical microscope: It appears as a distinct, visible edge.Incomplete sealing as with a rectangular channel appears as an “island”of contact in the flow channel; complete sealing (as observed withrounded channels) gives a continuous contact edge joining the left andright edges of the flow channel.

Optical micrographs of different valve and pump configurations; controllines are oriented vertically. (A) Simple on-off valve with 200-μmcontrol line and 100-μm flow line (“200×100”). (B) See FIG. 2 of“Monolithic Microfabricated Valves and Pumps by Multilayer SoftLithography”, Unger et al., Science, Vol. 288, pp. 113-116 (Apr. 7,2000) 30×50 on-off valve. (C) Peristaltic pump. Only three of the fourcontrol lines shown were used for actuation. (D) Grid of on-off valves.(E) Switching valve. Typically, only the innermost two control lineswere used for actuation. (F) Section of the seven-layer test structurementioned in the text. All scale bars are 200 μm.

(A) A 3D scale diagram of an elastomeric peristaltic pump. The channelsare 100 μm wide and 10 μm high. Peristalsis was typically actuated bythe pattern 101, 100, 110, 010, 011, 001, where 0 and 1 indicate “valveopen” and “valve closed,” respectively. This pattern is named the “120°”pattern, referring to the phase angle of actuation between the threevalves. Other patterns are possible, including 90° and 60° patterns. Thedifferences in pumping rate at a given frequency of pattern cycling wereminimal. (B) Pumping rate of a peristaltic micropump versus variousdriving frequencies. Dimension of microvalves=100 μm by 100 μm by 10 μm;applied air pressure=50 kPa.

The application of micromachining techniques is growing rapidly, drivenby the dramatic success of a few key applications such asmicrofabricated accelerometers (1, 2), pressure sensors (3), and ink-jetprint heads (4). New applications are appearing in other fields, inparticular fiber optic communications (5, 6), displays (7), andmicrofluidics (8-12). The two most widespread methods for the productionof microelectromechanical structures (MEMS) are bulk micromachining andsurface micromachining. Bulk micromachining is a subtractive fabricationmethod whereby single-crystal silicon is lithographically patterned andthen etched to form three-dimensional (3D) structures. Surfacemicromachining, in contrast, is an additive method where layers ofsemiconductor-type materials (polysilicon, metals, silicon nitride,silicon dioxide, and so forth) are sequentially added and patterned tomake 3D structures.

Bulk and surface micromachining methods are limited by the materialsused. The semiconductor-type materials typically used in bulk andsurface micromachining are stiff materials with Young's modulus ˜100 GPa(13). Because the forces generated by micromachined actuators arelimited, the stiffness of the materials limits the minimum size of manydevices. Furthermore, because multiple layers must be built up to makeactive devices, adhesion between layers is a problem of great practicalconcern. For bulk micromachining, wafer-bonding techniques must be usedto create multilayer structures. For surface micromachining, thermalstress between layers limits the total device thickness to ˜20 μm.Clean-room fabrication and careful control of process conditions arerequired to realize acceptable device yields.

An alternative microfabrication technique based on replication moldingis gaining popularity. Typically, an elastomer is patterned by curing ona micromachined mold. Loosely termed soft lithography, this techniquehas been used to make blazed grating optics (14), stamps for chemicalpatterning (15), and microfluidic devices (16-20). Soft lithography'sadvantages include the capacity for rapid prototyping, easy fabricationwithout expensive capital equipment, and forgiving process parameters.For applications with moderate-sized features (>20 μm) such asmicrofluidics, molds can be patterned by using a high-resolutiontransparency film as a contact mask for a thick photoresist layer (21).A single researcher can design, print, pattern the mold, and create anew set of cast-elastomer devices within 1 day, and subsequent elastomercasts can be made in just a few hours. The tolerant process parametersfor elastomer casting allow devices to be produced in ambient laboratoryconditions instead of a clean room. However, soft lithography also haslimitations: It is fundamentally a subtractive method (in the sense thatthe mold defines where elastomer is removed), and with only oneelastomer layer it is difficult to create active devices or movingparts. A method for bonding elastomer components by plasma oxidation hasbeen described previously (21) and has been used to seal microfluidicchannels against flat elastomer substrates (20,22).

We describe here a technique called “multilayer soft lithography” thatcombines soft lithography with the capability to bond multiple patternedlayers of elastomer. Multilayer structures are constructed by bondinglayers of elastomer, each of which is separately cast from amicromachined mold (FIGS. 1-4). The elastomer is a two-componentaddition-cure silicone rubber. The bottom layer has an excess of one ofthe components (A), whereas the upper layer has an excess of the other(B). After separate curing of the layers, the upper layer is removedfrom its mold and placed on top of the lower layer, where it forms ahermetic seal. Because each layer has an excess of one of the twocomponents, reactive molecules remain at the interface between thelayers. Further curing causes the two layers to irreversibly bond: Thestrength of the interface equals the strength of the bulk elastomer.This process creates a monolithic three-dimensionally patternedstructure composed entirely of elastomer. Additional layers are added bysimply repeating the process: Each time the device is sealed on a layerof opposite “polarity” (A versus B) and cured, another layer is added.

The ease of producing multilayers makes it possible to have multiplelayers of fluidics, a difficult task with conventional micromachining.We created test structures of up to seven patterned layers in thisfashion (23), each of ˜40 μm thickness (FIG. 2F). Because the devicesare monolithic (i.e., all of the layers are composed of the samematerial), interlayer adhesion failures and thermal stress problems arecompletely avoided. Particulates disturb interlayer adhesion verylittle, if at all. Perhaps most importantly for the actuation ofmicrostructures, the elastomer is a soft material with Young's modulus(24) ˜750 kPa, allowing large deflections with small actuation forces.One can also control the physical properties of the material. We createdmagnetic layers of elastomer by adding fine iron powder and electricallyconducting layers by doping with carbon black above the percolationthreshold (25). There is thus the possibility of creating all-elastomerelectro-magnetic devices (26).

To demonstrate the power of multilayer soft lithography, we fabricatedactive valves and pumps. Monolithic elastomeric valves and pumps, likeother mechanical microfluidic devices, avoid several practical problemsaffecting flow systems based on electroosmotic flow (8,9, 20, 27-29) ordielectrophoresis (30, 31), such as electrolytic bubble formation aroundthe electrodes and a strong dependence of flow on the composition of theflow medium (32-34). Electrolytic bubble formation, although not aproblem for laboratory devices, seriously restricts the use ofelectroosmotic flow in integrated microfluidic devices. Also, neitherelectroosmotic nor dielectrophoretic flow can easily be used to stopflow or balance pressure differences.

We fabricated our valves using a crossed-channel architecture (FIG. 1A).Typical channels are 100 μm wide and 10 μm high, making the active areaof the valve 100 μm by 100 μm. The membrane of polymer between thechannels is engineered to be relatively thin (typically 30 μm). Whenpressure is applied to the upper channel (“control channel”), themembrane deflects downward. Sufficient pressure closes the lower channel(“flow channel”). For optical convenience, we typically seal ourstructures with glass as the bottom layer; this bond with glass isreversible, so devices may be peeled up, washed, and reused. We alsofabricated devices where the bottom layer is another layer of elastomer,which is useful when higher back pressures are used. The response timeof devices actuated in this fashion is on the order of 1 ms, and theapplied pressures are on the order of 100 kPa, so a 100 μm by 100 μmarea gives actuation forces on the order of 1 mN. Pneumatic actuationallows active devices to be densely packed; we built microfluidics withdensities of 30 devices per square millimeter, and greater densities areachievable. This actuation speed, pressure, and device density are morethan adequate for the vast majority of microfluidic applications.

The shape of the flow channel is important for proper actuation of thevalve (FIG. 1B). Rectangular and even trapezoidal shaped channels willnot close completely under pressure from above. Flow channels with around cross section close completely; the round shape transfers forcefrom above to the channel edges and causes the channel to close fromedges to center. We found that 100 μm by 100 μm by 10 μm valves overtrapezoidal channels would not close completely even at 200 kPa ofapplied pressure, whereas rounded channels sealed completely at only 40kPa.

Making multiple, independently actuated valves in one device simplyrequires independent control of the pressure applied to each controlline (35). FIG. 2, A to E, shows simple configurations resulting inon-off valves (FIGS. 2, A and B), a pump (FIG. 2C), a grid of valves(FIG. 2D), and a switching valve (FIG. 2E). Each control line canactuate multiple valves simultaneously. Because the width of the controllines can be varied and membrane deflection depends strongly on membranedimensions, it is possible to have a control line pass over multipleflow channels and actuate only the desired ones. The active element isthe roof of the channel itself, so simple on-off valves (and pumps)produced by this technique have truly zero dead volume; switching valveshave a dead volume about equal to the active volume of one valve, thatis, 100 μm×100 μm×10 μm=100 pl. The dead volume required and the areaconsumed by the moving membrane are each about two orders of magnitudesmaller than any microvalve demonstrated to date (11).

The valve opening can be precisely controlled by varying the pressureapplied to the control line. As demonstrated in FIG. 21, the response ofthe valve is almost perfectly linear over a large portion of its rangeof travel, with minimal hysteresis. Thus, these valves can be used formicrofluidic metering and flow control. The linearity of the valveresponse demonstrates that the individual valves are well-modeled asHooke's law springs. Furthermore, high pressures in the flow channel(“back pressure”) can be countered simply by increasing the actuationpressure. Within the experimental range we were able to test (up to70-kPa back pressure), valve closing was achieved by simply adding theback pressure to the minimum closing pressure at zero back pressure.

(A) Valve opening versus applied pressure. “50x 100” indicates amicrovalve with a 50-μm-wide control channel and a 100-μm-wide fluidchannel. 100×50 closing and opening data (not shown) are nearlyidentical to 50×100 data. (B) Time response of a 100 μm by 100 μm by 10μm RTV microvalve with 10-cm-long air tubing connected from the chip toa pneumatic valve. Two periods of digital control signal, actual airpressure at the end of the tubing, and valve opening are shown here. Thepressure applied on the control line is 100 kPa, which is substantiallyhigher than the ˜40 kPa required to close the valve. Thus, when closing,the valve is pushed closed with a pressure 60 kPa greater than required.When opening, however, the valve is driven back to its rest positiononly by its own spring force (≦40 kPa). Thus, τ close is expected to besmaller than τ open. There is also a lag between the control signal andcontrol pressure response, due to the limitations of the miniature valveused to control the pressure. Calling such lags t and the 1/e timeconstants τ, the values are t open=3.63 ms, τ open=1.88 ms, t close=2.15ms, and τ close=0.51 ms. If 3 τ each are allowed for opening andclosing, the valve runs comfortably at 75 Hz when filled with aqueoussolution (36). Valve opening was measured by fluorescence. The flowchannel was filled with a solution of fluorescein isothiocyanate inbuffer (pH≧8), and the fluorescence of a square area occupying thecenter third of the channel was monitored on an epi-fluorescencemicroscope with a photomultiplier tube with a 10-kHz bandwidth. Thepressure was monitored with a Wheatstone-bridge pressure sensor(SCC15GD2; Sensym, Milipitas, Calif.) pressurized simultaneously withthe control line through nearly identical pneumatic connections.

Monolithic elastomer valves fabricated as described here can be actuatedwith surprising speed. The time response for a valve filled with aqueoussolution is on the order of 1 ms, as shown in FIG. 22. The valve stillopens and closes at 100 Hz, although it does not open completely. Thevalve responds nearly instantaneously to the applied pressure, butapplied pressure lags substantially behind the control signal (36).

We also fabricated a peristaltic pump from three valves arranged on asingle channel (FIG. 26, A-B). Pumping rates were determined bymeasuring the distance traveled by a column of water in thin (0.5 mminterior diameter) tubing; with 100 μm by 100 μm by 10 μm valves, amaximum pumping rate of 2.35 nl/s was measured (FIG. 25). Consistentwith the previous observations of valve actuation speed, the maximumpumping rate is attained at ˜75 Hz; above this rate, increasing numbersof pump cycles compete with incomplete valve opening and closing. Thepumping rate was nearly constant until above 200 Hz and fell off slowlyuntil 300

Hz. The valves and pumps are also quite durable: We have never observedthe elastomer membrane, control channels, or bond to fail. None of thevalves in the peristaltic pump described above show any sign of wear orfatigue after more than 4 million actuations. In addition to theirdurability, they are also gentle. A solution of Escherichia coli pumpedthrough a channel and tested for viability showed a 94% survival rate(37).

Monolithic active valves built as described here have several notableadvantages over silicon-based microfluidic valves. Because of the lowYoung's modulus of silicone rubber, the valves' active area is no largerthan the channels themselves; this permits exceptionally low deadvolumes. Because of the softness of the membrane, complete valve sealingis easily attained, even in the presence of particulates. The valvesclose linearly with applied pressure, allowing metering and permittingthem to close in spite of high back pressure. Their small size makesthem fast, and size and softness both contribute to making them durable.Small size, pneumatic actuation, and the ability to cross channelswithout actuating them allow a dense integration of microfluidic pumps,valves, mixing chambers, and switch valves in a single,easy-to-fabricate microfluidic chip. The greatest advantage, however, isease of production. Compared with valves and pumps made withconventional silicon-based micromachining (11) [or even hybrid devicesincorporating polymers (38-41)], monolithic elastomer valves are simplerand much easier to fabricate.

The use of nontraditional materials gives the multilayer softlithography method a number of advantages over conventionalmicromachining, including rapid prototyping, ease of fabrication, andforgiving process parameters. It allows multilayer fabrication withoutthe problems of interlayer adhesion and thermal stress buildup that areendemic to conventional micromachining. This process can be used toconstruct complex multilayer microfabricated structures such as opticaltrains and microfluidic valves and pumps. The silicone rubber used hereis transparent to visible light, making optical interrogation ofmicrofluidic devices simple. It is also biocompatible—materials in thisfamily are used to fabricate contact lenses. The raw material isinexpensive, especially when compared with single-crystal silicon(˜$0.05/cm3 compared with ˜$2.5/cm3). Most important, it has a lowYoung's modulus, which allows actuation even of small area devices.Pneumatically actuated valves and pumps will be useful for a widevariety of fluidic manipulation for lab-on-a-chip applications. In thefuture, it should be possible to design electrically or magneticallyactuated valves and pumps that can be used as implantable devices forclinical applications.

-   1. L. M. Roylance et al., IEEE Trans. Electron Devices ED-26, 1911    (1979)-   2. N. Yazdi et al., Proc. IEEE 86, 1640 (1998)-   3. O. N. Tufte et al., J. Appl. Phys. 33, 3322 (1962)-   4. L. Kuhn et al., IEEE Trans. Electron Devices ED-25, 1257 (1978)-   5. L. Y. Lin et al., IEEE J. Selected Top. Quantum Electron. 5, 4    (1999)-   6. R. S. Muller et al., Proc. IEEE 86, 1705 (1998)-   7. L. J. Hornbeck et al., OSA Tech. Dig. Ser. 8, 107 (1988)-   8. D. J. Harrison et al., Science 261, 895 (1993)-   9. S. C. Jacobson et al., Anal. Chem. 66, 1114 (1994)-   10. M. U. Kopp et al., Science 280, 1046 (1998)-   11. S. Shoji, Top. Curr. Chem. 194, 163 (1998)-   12. P. Gravesen et al., Microeng. 3, 168 (1993)-   13. 1. L. Buchaillot et al., Jpn. J. Appl. Phys. 2 36, L794 (1997)-   14. Y. N. Xia et al., Science 273, 347 (1996)-   15. Y. N. Xia et al., Angew. Chem. Int. Ed. Engl. 37, 550 (1998)-   16. C. S. Effenhauser et al., Anal. Chem. 69, 3451 (1997)-   17. E. Delamarche et al., Science 276, 779 (1997)-   18. A. Y. Fu et al., Nature Biotechnol. 17, 1109 (1999)-   19. K. Hosokawa et al., Anal. Chem. 71, 4781 (1999)-   20. D. C. Duffy et al., J. Micromech. Microeng. 9, 211 (1999)-   21. D. C. Duffy et al., Anal. Chem. 70, 4974 (1998)-   22. P. J. A. Kenis et al., Science 285, 83 (1999).-   23. For multilayers, a thick layer was prepared as previously    described; each thin layer was baked at 80° C. for 20 min. The    growing thick layer was assembled on each new thin layer and bonded    by baking at 80° C. for 20 min. Seven-layer devices have been    produced by this method; no obvious limitations exist to limit the    number of layers.-   24. J. C. Lötters et al., J. Micromech. Microeng. 7, 145 (1997)-   25. Conductive silicone was created by the addition of a fine carbon    black (Vulcan XC72; Cabot, Billerica, Mass.) at 10% or higher    concentration by weight. Conductivity increased with carbon black    concentration from 5.6×10-16 to ˜5×10-3 (ohm·cm)−1. Magnetic    silicone was created by the addition of iron powder (˜1 μm particle    size); up to 20% Fe by weight was added. For both conductive and    magnetic silicones, multilayer bonding functioned normally.-   26. K. Ikuta, K. Hirowatari, T. Ogata, in Proceedings IEEE    International MEMS 94 Conference (IEEE, Piscataway, N.J., 1994), pp.    1-6.-   27. R. B. M. Schasfoort et al., Science 286, 942 (1999)-   28. S. C. Jacobson et al., Anal. Chem. 71, 4455 (1999)-   29. C. S. Effenhauser et al., Electrophoresis 18, 2203 (1997)-   30. M. Washizu et al., IEEE Trans. Ind. Appl. 30, 835 (1994)-   31. R. Pethig et al., Trends Biotechnol. 15, 426 (1997)-   32. R. Brechtel et al., J. Chromatogr. A 716, 97 (1995-   33. C. A. Lucy et al., Anal. Chem. 68, 300 (1996)-   34. The magnitude of flow (and even its direction) depends in a    complicated fashion on ionic strength and type, the presence of    surfactants, and the charge on the walls of the flow channel;    furthermore, because electrolysis is taking place continuously, the    capacity of buffer to resist pH changes is finite. Precise control    of flow thus requires calibration for each new buffer or solute and    can be difficult when the exact composition of a sample is not known    in advance. Electroosmotic flow can also induce unwanted    electrophoretic separation of molecules, creating demixing problems.    Dielectrophoresis does not require electrolysis and therefore does    not cause bubble formation but still suffers from sample and solvent    sensitivity.-   35. Each control channel was connected to the common port of a    miniature three-way switch valve (LHDA1211111H; Lee Valve,    Westbrook, Conn.), powered by a fast Zener-diode circuit and    controlled by a digital data acquisition card (AT-DIO-32HS; National    Instruments, Austin, Tex.). Regulated external pressure was provided    to the normally closed port, allowing the control channel to be    pressurized or vented to atmosphere by switching the miniature    valve.-   36. If one used another actuation method that did not suffer from    opening and closing lag, this valve would run at ˜375 Hz. The spring    constant can be adjusted by changing the membrane thickness; this    allows optimization for either fast opening or fast closing.-   37. E. coli were pumped at 10 Hz through the channel. Samples of    known volume were taken from the output well (pumped) and the input    well (control), and serial dilutions of each were plated on    Luria-Bertani agar plates and grown overnight at 37° C. Viability    was assessed by counting colonies in the control and pumped samples    and correcting for sample volumes and dilution.-   38. J. Fahrenberg et al., J. Micromech. Microeng. 5, 77 (1995)-   39. C. Goll et al., J. Micromech. Microeng. 6, 77 (1996)-   40. X. Yang et al., Sensors Actuators A 64, 101 (1998)-   41. A. M. Young et al., J. Biomech. Eng. Trans. ASME 121, 2 (1999)

While the present invention has been described herein with reference toparticular embodiments thereof, a latitude of modification, variouschanges and substitutions are intended in the foregoing disclosure, andit will be appreciated that in some instances some features of theinvention will be employed without a corresponding use of other featureswithout departing from the scope of the invention as set forth.Therefore, many modifications may be made to adapt a particularsituation or material to the teachings of the invention withoutdeparting from the essential scope and spirit of the present invention.It is intended that the invention not be limited to the particularembodiment disclosed as the best mode contemplated for carrying out thisinvention, but that the invention will include all embodiments andequivalents falling within the scope of the claims.

The following pending patent applications contain subject matter relatedto the instant application and are hereby incorporated by reference:U.S. application Ser. No. 09/970,122, now U.S. Pat. No. 7,258,374; andU.S. application Ser. No. 09/724,548, now U.S. Pat. No. 7,351,376.

1. (canceled)
 2. The method of using a microfluidic device, comprising:flowing a sample into a looped channel of the microfluidic device;pumping the sample around the loop channel with a peristaltic action,wherein the peristaltic action comprises sequential activation of aseries of individually addressable valves arranged along the loopedchannel.
 3. The method of claim 2, wherein the series of individuallyaddressable valves comprise three valves.
 4. The method of claim 3,wherein each of the three valves is an intersection of an individuallyaddressable control line over the looped channel.
 5. The method of claim2, wherein the microfluidic device is an elastomeric device.
 6. Themethod of claim 2, wherein the pumping passes the sample over a DNAarray.